In vitro determination of the CYP 3A4 activity in rat hepatic microsomes by liquid-phase extraction and HPLC-photodiode array detection.

INTRODUCTION: CYP3A4 is one of the most important of all drug-metabolizing enzymes. Although several direct or indirect quantification methods have been proposed for the determination of the CYP3A4 activity, the sample preparation is mostly tedious and usually requires time consuming separate extraction steps and solid-phase extraction. METHODS: Here, we developed a simple and selective HPLC method, coupled to photodiode array detection for direct determination of CYP3A4-mediated testosterone hydroxylase activity in hepatic microsomes of rats. After microsome incubation, a single-step liquid-phase extraction of specific substrates, testosterone and its metabolite 6-hydroxytestosterone, together with the salicylamid acid used as an internal standard, was applied. The analytical method was fully validated by the determination of different parameters (intra- and inter-day variability of 6-ß-OH-testosterone concentration, accuracy and limit of detection). Finally, this method was applied to quantify the CYP3A4 activity of rats intravenously administered with either the mesoporous iron(III) trimesate MIL-100 nanocarrier (MIL stands for Materials from Institut Lavoisier) or with its corresponding organic linker. RESULTS: All analytes were simultaneously separated from a single run shorter than 10 min, reaching relative standard deviations of intra- and inter-day precision <18.5% and an accuracy of estimated 6-ß-OH-testosterone concentrations ranging from 95 to 111%. The mean±standard deviation absolute recoveries of 6-ß-OH-testosterone at 0.01, 1.00 and 20.00 µg/mL were 97±4%, 101±3% and 99±2%, respectively while it reached 98±3% for the internal standard. DISCUSSION: The developed HPLC-PDA method enables the accurate and sensitive determination of the CYP3A4 activity through the quantification of the 6-ß-OH-testosterone produced by the CYP3A4-mediated testosterone hydroxylase in rat hepatic microsomal suspensions. Additionally, the rapid procedure offers an economical advantage with respect to resources and operator time. Finally, the determination of CYP3A4 activity in hepatic microsomes of rats administered with nanoparticles of the porous iron(III) trimesate nanocarrier shows no significant differences between control, trimesic and nanoparticle groups, evidencing that the linker is not metabolized by CYP3A4.

Rapid quantitation of testosterone hydroxyl metabolites by ultra-performance liquid chromatography and mass spectrometry.

A rapid and sensitive ultra-performance liquid chromatography and mass spectrometry (UPLC/MS) method was developed to simultaneously quantify seven monohydroxyl testosterone metabolites (16alpha-, 2alpha-, 7alpha-, 6alpha-, 2beta-, 6beta-, and 16beta-hydroxyl testosterones) in rat liver microsomes. The UPLC system used a short 1.7-microm particle size column coupled to a Sciex 4000 Q trap in multiple reaction monitor (MRM) mode. All hydroxyl testosterones were resolved within 2.5 min. A 4-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method in rat liver microsomes. This method is applicable to the measurement of the testosterone hydroxylase activity in biological matrices such as the liver microsome incubates.

High-throughput cytochrome P450 inhibition assays using laser diode thermal desorption-atmospheric pressure chemical ionization-tandem mass spectrometry.

This paper describes the development of a high-throughput method for the analysis of cytochrome P450 (CYP) inhibition assay incubation samples using laser diode thermal desorption interfaced with atmospheric pressure chemical ionization mass spectrometry (LDTD-APCI-MS). Data for the CYP isoforms 3A4, 2D6, 2C9, and 1A2 from competitive inhibition assays are shown. The potential for inhibition of the CYP isoforms was measured by monitoring the level of the metabolites 6beta-hydroxytestosterone (3A4), dextrorphan (2D6), 4'-hydroxydiclofenac (2C9), and acetaminophen (1A2) formed in the presence of drug candidates using an eight-point titration. The analytical method involves plating of the inhibition samples on specially designed 96-well plates with stainless steel bottoms, followed by direct analysis using the LDTD source. Validation of the LDTD-MS method was performed by testing for interferences, reproducibility, dynamic range, ion suppression, and the ability of the source to produce comparable results to previously validated LC-MS methods. IC50 values for each CYP isoform using 33 different test compounds showed excellent agreement between LDTD-APCI-MS and LC-MS methods and literature values where available. Assay analysis time using the LDTD-APCI source is reduced to less than 30 min for a single 96-well plate compared to greater than 10 h using the LC-MS method. The LDTD-APCI-MS and LC-MS methods and results are compared and limitations and future potential for LDTD-APCI-MS are discussed.

The use of heat treatment to eliminate drug interactions due to grapefruit juice.

Grapefruit juice (GJ) contains components that may increase the bioavailability of drugs; however, approaches to the removal of these components have been little investigated. It is known that furanocoumarin derivatives (FCs), such as bergamottin (BG) and 6',7'-dihydroxybergamottin (DHB) in GJ, induce such drug interactions. In the present study, it was found that the heat treatment of grapefruit juice decreases concentrations of BG and DHB as well as their interactions both in vitro and in vivo. We incubated GJ for 10, 20, 30, 40, 50, and 60 min at 37, 62, 72, and 95 degrees C; FCs in each sample were then measured, using high-performance liquid chromatography (HPLC). The concentrations of BG and DHB were decreased in a time- and temperature-dependent manner, by 82.5 and 97.9% respectively, after incubation for 1 h at 95 degrees C. In contrast, the concentration of bergaptrol (BT) increased in a time- and temperature-dependent manner (27.7% after 60 min at 95 degrees C). In addition, the effect of each GJ sample on testosterone 6beta-oxidation in human liver microsomes was observed. The inhibitory effects of GJ heated to 95 degrees C were decreased in a time-dependent manner, as in the case of BG and DHB concentrations. Furthermore, 2 ml of GJ treated for 60 min at 95 degrees C was administered into the rat duodenum. After 30 min, nifedipine (NFP) was administered intraduodenally at a dose of 3 mg/kg body weight. The concentrations of NFP in the plasma samples were determined by HPLC. No significant increase in the AUC of NFP was observed in the rats given heat-treated GJ. These results suggest that the heat treatment of GJ reduces the concentrations of FCs, thus eliminating the potential for drug interactions.

Comparison of synthesis of 15 alpha-hydroxylated steroids in males of four North American lamprey species.

Recent studies have provided evidence that 15 alpha-hydroxytestosterone (15 alpha-T) and 15 alpha-hydroxyprogesterone (15 alpha-P) are produced in vitro and in vivo in adult male sea lampreys (Petromyzon marinus), and that circulatory levels increase in response to injections with gonadotropin-releasing hormone (GnRH). We examined four species from the Petromyzontidae family including silver lampreys (Ichthyomyzon unicuspis), chestnut lampreys (I. castaneus), American brook lampreys (Lethenteron appendix), and Pacific lampreys (Entosphenus tridentatus) to determine if these unusual steroids were unique to sea lampreys or a common feature in lamprey species. In vitro production was examined through incubations of testis with tritiated precursors, and 15 alpha-T and 15 alpha-P production was confirmed in all species through co-elution with standards on both high performance liquid chromatography (HPLC) and thin layer chromatography. In vivo production was proven by demonstrating that HPLC-fractionated plasma had peaks of immunoreactive 15 alpha-T and 15 alpha-P that co-eluted with standards through using previously developed radioimmunoassays for 15 alpha-T and 15 alpha-P. The possible functionality of 15 alpha-T and 15 alpha-P was further examined in silver and Pacific lampreys by investigating the effect of injection of either type of lamprey GnRH on plasma concentrations of 15 alpha-T and 15 alpha-P. Injections with exogenous GnRH did not affect circulatory levels of either steroid in silver lampreys, and only GnRH III elicited higher levels of both steroids in Pacific lampreys. The 15 alpha-hydroxylase enzyme(s) for steroids appeared to present in adult males of all species examined, but the question of whether 15 alpha-hydroxylated steroids are functional in these lamprey species, and the significance of the 15-hydroxyl group, requires further research.

Development and validation of a high-throughput radiometric CYP3A4/5 inhibition assay using tritiated testosterone.

A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 3A4/5 in human liver microsomes is described. In contrast to the conventional testosterone 6beta-hydroxylation assay, the new method does not require high-performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP3A4/5-mediated 6beta-hydroxylation of testosterone labeled with tritium in the 6beta position. The radiolabeled product is separated from the substrate using 96-well solid-phase extraction plates. Using commercially available [1,2,6,7-(3)H]testosterone as substrate, we demonstrated that the reaction is NADPH-dependent, and sensitive to CYP3A4/5/5 inhibitors and a CYP3A4/5/5-specific inhibitory monoclonal antibody, but not to inhibitors of or antibodies against other P450 enzymes. The method was further improved by synthesis of testosterone specifically tritiated in the 6beta position, which displayed greatly improved conversion rate with an ensuing increase in assay sensitivity. Competition experiments using tritiated and unlabeled testosterone indicated that CYP3A4/5-mediated 6beta-hydroxylation exhibits positive cooperativity and a modest kinetic isotope effect. IC(50) values for more than 40 structurally diverse chemical inhibitors were not significantly different from those determined in the testosterone 6beta-hydroxylation assay, using HPLC-tandem mass spectrometry analysis. All the steps of the new assay, namely, incubation, product separation, and radioactivity counting, are performed in 96-well format and can be automated. This assay thus represents a high-throughput version of the classical testosterone 6beta-hydroxylation assay, which is the most widely used method to assess the potential for CYP3A4/5 inhibition of new chemical entities.

Quantitative analysis of eight testosterone metabolites using column switching and liquid chromatography/tandem mass spectrometry.

The rate at which testosterone is metabolized to different singly hydroxylated metabolites has been widely used as an in vitro marker for activity of different CYP450 enzymes. The interest in extra-hepatic metabolism, e.g. due to metabolism in the gut wall, has increased during the last decade. Measurement of extra-hepatic enzyme activity using testosterone as a substrate requires a highly sensitive analytical method. A new liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) method, using column switching for online cleaning and desalting of samples, was developed and validated for analysis of 2alpha-, 2beta-, 6alpha-, 6beta-, 7alpha-, 16alpha-, and 16beta-hydroxytestosterone and androstenedione. The samples were injected on a SB-CN column and detection was performed using MS/MS. The limits of quantification ranged from 0.3 to 3.33 nM for the different metabolites. The validated method was used to quantify the enzyme activity in rat intestine mucosa. The formation rates of 16alpha-, 16beta-hydroxytestosterone and androstenedione were quantified, and 2beta-and 6beta-hydroxytestosterone were formed above the limits of detection.

15 alpha-Hydroxytestosterone produced in vitro and in vivo in the sea lamprey, Petromyzon marinus.

Prior research has shown that the testes of lampreys are able to synthesize 15-hydroxylated steroid hormones in vitro. Here we show that testes of the sea lamprey Petromyzon marinus L. are able to convert tritiated testosterone into tritiated 15alpha-hydroxytestosterone (15alpha-T) in high yield. The identity of the tritiated 15alpha-T has been confirmed by: co-elution with standard 15alpha-T on high performance liquid chromatography (HPLC); co-elution on thin layer chromatography (TLC); co-elution of acetylated tritiated and standard 15alpha-T on TLC; and strong binding to an antiserum developed against 15alpha-T. The strong reaction between the tritiated 15alpha-T and the antiserum has been used to develop a radioimmunoassay (RIA). The RIA operates over the range of 500-2pg per tube; and can be applied directly to plasma samples. This assay has been used to demonstrate that 15alpha-T is present in blood plasma of the sea lamprey. The concentrations of 15alpha-T in captive lamprey were found to be as follows (pg/ml; mean+/-SEM, n): parasitic stage (reproductively immature), <20, n=7; pre-ovulatory females, 156+/-30, n=8; ovulated females, 62+/-9, n=5; pre-spermiating males, 275+/-19, n=8; spermiating males, 216+/-48, n=8. When spermiating male plasma was fractionated on HPLC, immunoreactivity was found exclusively in the expected elution position of 15alpha-T. The biological significance of this steroid has yet to be established.

Studies on Bacillus stearothermophilus. Part III. Transformation of testosterone.

Bacillus stearothermophilus, a thermophilic bacterium isolated from the Kuwaiti desert, produced a variety of monohydroxy androstene derivatives and an oxidized product when incubated with exogenous testosterone for 24 h at 65 degrees C. The major metabolite was identified as androst-4-en-3,17-dione while minor metabolites included 6 alpha-hydroxyandrost-4-en-3,17-dione, 6 beta-hydroxyandrost-4-en-3,17-dione, 6 alpha-hydroxytestosterone, and 6 beta-hydroxytestosterone. These metabolites were purified by TLC and HPLC followed by their identification using (1)H- and (13)C-NMR and other spectroscopic data.

Changes in the metabolic elimination profile of testosterone following exposure of the crustacean Daphnia magna to tributyltin.

The biocide tributyltin has been found to cause the development of pseudohermaphroditic conditions in some neogastropod species. These abnormalities of the reproductive system have adversely affected the fecundity of some field populations of gastropods, resulting in local population declines. Current evidence suggests that tributyltin elicits these effects by interfering with the biotransformation of testosterone to other steroid derivatives, resulting in an elevation in endogenous testosterone or some of its bioactive derivatives. The purpose of the present study was to determine whether tributyltin altered testosterone metabolism in daphnids (Daphnia magna), a species commonly used in ecotoxicology testing. Exposure of daphnids to 1.2 microg (tin)/L caused a general increase in the rate of elimination of oxido-reduced, hydroxylated, and glucose-conjugated derivatives of testosterone. However, tributyltin exposure had no significant effect on the rate of elimination of the glucose-conjugated forms of the various oxido-reduced and hydroxylated derivatives of testosterone. As a result, the percentage of the oxido-reduced and hydroxylated metabolites of testosterone eliminated as glucose conjugates decreased with increasing tributyltin exposure levels. These results demonstrate that tributyltin causes alterations in testosterone metabolism in daphnids that would result in an increase in the production of oxido-reduced derivatives. These products are preferentially retained in the tissues of daphnids and are variously androgenic in vertebrates. The increased production of oxido-reduced derivatives of testosterone may be mechanically responsible for the masculinizing effects of tributyltin in some species and suggests that daphnids may be a suitable surrogate for evaluating the potential of chemicals to elicit this form of toxicity.

Determination of testosterone and 6beta-hydroxytestosterone by gas chromatography-selected ion monitoring-mass spectrometry for the characterization of cytochrome p450 3A activity.

A method for the determination of testosterone and its metabolite, 6beta-hydroxytestosterone, in liver microsomal incubates employing gas chromatography with selected ion monitoring mass spectrometric detection (GC-SIM-MS) has been developed. The method is more rapid than previously reported methods. Testosterone and its metabolites are extracted from the incubation mixture in a single step with methylene chloride. The method does not require derivatization and testosterone and its metabolites are separated on a HP-5MS fused-silica capillary column in less than 15 min. The retention times of testosterone (m/z 288), methyltestosterone (m/z 302), and 6beta-hydroxytestosterone (m/z 304) are approximately 12.7, 12.8, and 13.4 min, respectively. There are no interferences from other known CYP450 metabolites of testosterone. In addition, the selectivity and specificity of the mass spectrometer helps eliminate possible interferences from drugs and new chemical entities evaluated using this methodology. Calibration curves for testosterone and 6beta-hydroxytestosterone are linear from 0.25 to 100 microM. Extraction recoveries are better than 92% for both analytes and the internal standard, methyltestosterone. Over the course of five separate runs, within-day and inter-day precision (expressed as relative standard deviation) was less than 5% for all concentrations of testosterone and 6beta-hydroxytestosterone. Accuracies ranged from 95.8 to 105.8% for testosterone and 94.6 to 104.2% for 6beta-hydroxytestosterone. The assay has been used to characterize the CYP3A metabolic activity of multiple preparations of human, rat, and dog liver microsomes.

Interindividual differences in hepatic expression of CYP3A4: relationship to genetic polymorphism in the 5'-upstream regulatory region.

Cytochrome P450 3A4, the most abundant P450 form in human liver, exhibits a very broad substrate specificity and is of great importance for drug metabolism. The interindividual difference in the hepatic expression of CYP3A4 is considerable. In order to investigate possible genetic factor(s) causing this variation, the rate of 6beta-hydroxylation of testosterone in human liver microsomes prepared from 46 different human liver samples was determined and the 5'upstream region (+10 to -490 bp) was sequenced from genomic DNA isolated from 39 of these livers. We found a 31-fold variation of the testosterone hydroxylase activity between the samples. However, a very high sequence homology between the CYP3A4 5'-upstream regions sequenced from the 78 different alleles was found. In fact, only three variant nucleotide exchanges were identified, all causing a -290 A-->G mutation (CYP3A4-V) in a so called nifedipine specific element (NFSE). The importance of this element and the polymorphism was evaluated by gel shift analysis. Competition experiments revealed that the binding of nuclear proteins, although having lower affinity to the CYP3A4-V form of the element, was unspecific in nature. In accordance, no influence of this polymorphism was seen on the microsomal testosterone hydroxylase activity in vitro. It is concluded that the promoter region of CYP3A4 is highly conserved, the only polymorphism being in the NFSE, which however does not influence the enzyme expression in liver to a significant degree. This casts doubt of a previously described relationship between the CYP3A4-V allele and cancer in the prostate and leukaemia.

Steroids and plasminogen activator concentrations in follicular fluid of gilts at first and third estrus.

An experiment was conducted to determine whether morphological and functional characteristics of follicles differed at a similar stage of pubertal (first) and third estrus in the same gilts. Nine prepubertal gilts were checked three times daily for estrus and laparotomized 6 h after detected first and third estrus. Samples of vena cava and ovarian venous blood were collected, follicle numbers and diameters were recorded, and follicular fluid (FF) was aspirated from all follicles 8 to 12 mm in diameter. Sera and(or) FF were analyzed for progesterone (P4), estradiol-17 beta (E2), testosterone (T), androstenedione (A4), 5 alpha-dihydrotestosterone (DHT), plasminogen activator (PA), and plasmin (PLM). Overall mean number of follicles > or = 8 mm in diameter did not differ between gilts at first and third estrus (P > .05) but gilts at first estrus had more follicles 4 to 8 (P < .05) and 8.1 to 10 mm in diameter (P < .01) and fewer 10.1 to 12 mm in diameter (P < .07) than at third estrus. Mean FF concentrations of E2, T, and A4 at third estrus were significantly greater than at first estrus, whereas FF concentrations of P4, DHT, PA, and PLM were similar at first and third estrus (P > .05). Mean concentrations of E2 in systemic and ovarian venous sera were also greater in gilts at third than at first estrus (both P < .05). Systemic concentrations of P4 in gilts at first and third estrus did not differ (P > .05).(ABSTRACT TRUNCATED AT 250 WORDS)

The identification of 14 alpha,17 beta-dihydroxyandrost-4-en-3-one monohydrate and 14 alpha,17 beta-dihydroxyandrosta-1,4-dien-3-one monohydrate, metabolites of androstenedione in Mucor piriformis.

The microorganism Mucor piriformis transforms androst-4-ene-3,17-dione into a major and several minor metabolites. X-ray crystallographic analysis of two of these metabolites was undertaken to determine unambiguously their composition and chirality. Crystals belong to the orthorhombic space-group P2(1)2(1)2(1), with a = 7.199(4) A and a = 6.023(3) A, b = 11.719(3) A and b = 13.455(4) A, c = 20.409(3) A and c = 20.702(4) A for the two title compounds, respectively. The structures have been refined to final R values of 0.060 and 0.040, respectively.

7-Alpha-hydroxytestosterone in seminiferous tubules of rat testis.

Production of 4-androstene-7 alpha,17 beta-diol-3-one (7 alpha-hydroxytestosterone) from testosterone was measured in seminiferous tubule and interstitial fractions of rat testis. In adults, specific activity of the 7 alpha-hydroxylase was about 10 times higher in interstitial cells than in seminiferous tubules, but tubule production was a significant portion of the total. Seminiferous tubule 7 alpha-hydroxylase was not detectable in weanlings. Also, we confirmed that 7 alpha-hydroxytestosterone inhibits the activity of the 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase active on testosterone and suggest a role in maturational changes.

[Distribution and metabolism of testosterone in rats]. / Studio sulla distribuzione e sul metabolismo del testosterone nel ratto.

Distribution and metabolism of testosterone were studied in male and female rats. While no differences between sexes were observed in the hormone distribution to liver, kidney, spleen, heart, adipose tissue, muscle, brain and adrenal, male liver was found to metabolize testosterone mainly to polar compounds; female liver mainly to A-ring reduced compounds.