Transformations of steroids and the steroidal alkaloid,solanine, by Phytophthora infestans

The following steroids and steroidal alkaloids have been incubated with the blight fungus Phytophthora infestans: androst-4-ene-3,17-dione, cholesterol, cholesteryl acetate, cholesteryl myristate, cholesteryl palmitate,cholesteryl stearate, dehydroisoandrosterone, 6α-hydroxy-androst-4-ene-3,17-dione, 6β-hydroxyandrost-4-ene-3,17-dione, 11α-hydroxyprogesterone, pregnenolone, progesterone, sitosterol, sitosteryl acetate, solanidine, solanine, stigmasterol, stigmasteryl acetate and testosterone. No hydroxylation was observed, but the fungus is able to oxidize alcohol functions at C-3β, C-6α, C-11β and C-17β to carbonyl. In addition, hydrolysis of acetate to hydroxyl at C-3β, and of solanine to solanidine, was observed. The relationship between metabolism and the nature of substitution at C-17β is discussed.     

Progesterone transformation as a biochemical aid in classification of the genusEmericella

A total of 65 isolates representing 13Emericella taxa (5 isolates of each of 12 species and 1 variety) had the ability to transform progesterone into 11α-hydroxyprogesterone. A systematic variation could be observed between the different testedEmericella taxa with respect to the transformation products. The isolates were divided according to the transformation types into six groups: (1) progesterone was hydroxylated into 6β-hydroxyprogesterone, 11α-hydroxyprogesterone and 6β, 11α-dihydroxyprogesterone— found inEmericella acristata andE. dentata; (2) E. aurantio-brunna andE. parvathecia hydroxylated progesterone into 11α-hydroxyprogesterone, 17α-hydroxyprogesterone and 11α,17α-dihydroxyprogesterone; (3)E. nidulans andE. quadrilineata formed the same three products as members of group (2) and form epicortisol; (4)E. nidulans var.lata, E. bicolor andE. variecolor transformed progesterone into a mixture of mono- di-and trihydroxy products; (5)E. striata andE. sublata exhibited an oxidative splitting of the progesterone side chain in position C-17 and hydroxylated progesterone into mono-and dihydroxy products; (6)E. rugulosa andE. unguis had the ability to degrade progesterone side-chain and to hydroxylate it into mono-, di- and trihydroxy products. This biochemical differentiation may supplement the morphological and other biochemical criteria used in the classification of theEmericella taxa.     

Biotransformation of progesterone to 14 alpha-hydroxypregna-1,4-diene-3,20-dione, a novel fungal metabolite, by Colletotrichum antirrhini

The fermentation of progesterone by Colletotrichum antirrhini SC 2144 was examined. Instead of 15 alpha-hydroxyprogesterone, the reported product, this fungus converted progesterone to androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione, 14 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 11 alpha-hydroxypregn-4-ene-3,20-dione, 14 alpha-hydroxypregn-4-ene-3,20-dione, and a hitherto undescribed compound, 14 alpha-hydroxypregna-1,4-diene-3,20-dione.     

Specific antisera suitable for solid-phase radioimmunoassay of 11beta-hydroxyandrost-4-ene-3,17-dione

Specific antiserum has been developed for use in measuring 11β-hydroxyandrost-4-ene-3, 17-dione by radioimmunoassay (RIA). Rabbit antiserum was generated by employing the conjugate prepared by coupling 6β,11β-dihydroxyandrost-4-ene-3,17-dione 6-hemisuccinate with bovine serum albumin. The antiserum bound 68% of 50 picograms of 11β-hydroxyandrost-4-ene-3,17-dione-[1,2,6,7-³H] during characterization at a dilution of 1:12,500. Among the numerous steroids tested for cross-reactivity, 5α-androstane-3,17-dione, androst-4-ene-3,17-dione, and 11β-hydroxy-5α-androstane-3, 17-dione showed 2%, 5%, and 30% cross-reactivity respectively. The Rivanol-treated antiserum was coupled to Enzacryl AA, in order to study the feasibility of a solid-phase RIA, and this complex showed 50% binding with the labeled antigen at a dilution of 1:3000. The complex retained high specificity and should prove useful in a simple solid-phase RIA.     

Studies on the Microbiological Transformation of Steroids

An attempt was made to clarify how Pellicularia filamentosa f. sp. microsclerotia IFO 6298 capable of hydroxylating C₂₁-steroids at the C-19 position converts C₁₉-steroids, especially monohydroxyderivatives of androst-4-ene-3, 17-dione. Such substrates as 11β-hydroxyandrost-4-ene-3,17-dione (I), androst-4-ene-3, 11, 17-trione (II), androsta-1,4-diene-3, 17-dione (III), 11β-hydroxyandrosta-1,4-diene-3,17-dione (IV), 14α-hydroxyandrost-4-ene-3, 17-dione (V), 15α-hydroxyandrost-4-ene-3, 17-dione (VI) and 9α-hydroxyandrost-4-ene-3, 17-dione (VII) were converted by the organism. All the main and several minor products were then isolated and identified. As a result it is concluded that this organism converts I and II into 14α-hydroxyandrost-4-ene-3,11,17-trione, III and IV into 14α-hydroxyandrosta-1,4-diene-3,1l,17-trione, V into 11α 14α dihydroxyandrost-4-ene-3, 17-dione (main) and 11β, 14α-dihydroxyandrost-4-ene-3, 17-dione (minor, a tentative structure), VI into 11β, 15α-dihydroxyandrost-4-ene-3,17-dione (main) and 15α-hydroxyandrost-4-ene-3,11,17-trione (minor, a tentative structure) and VII into 9α, 14α-dihydroxyandrost-4-ene-3, 17-dione (main) and 6β, 9α-dihydroxyandrost-4-ene-3,17-dione (minor).

In addition, the structural requirement of substrate for the 19-hydroxylation catalyzed by the organism and the influence of a hydroxyl group on steroid nucleus upon the 11β- and 14α-hydroxylations and the 11β-OH-dehydrogenation was discussed.     

Biotransformation of dehydroepiandrosterone with Macrophomina phaseolina and β-glucuronidase inhibitory activity of transformed products

The biotransformation of dehydroepiandrosterone (1) with Macrophomina phaseolina was investigated. A total of eight metabolites were obtained which were characterized as androstane-3,17-dione (2), androst-4-ene-3,17-dione (3), androst-4-ene-17β-ol-3-one (4), androst-4,6-diene-17β-ol-3-one (5), androst-5-ene-3β,17β-diol (6), androst-4-ene-3β-ol-6,17-dione (7), androst-4-ene-3β,7β,17β?triol (8), and androst-5-ene-3β,7α,17β-triol (9). All the transformed products were screened for enzyme inhibition, among which four were found to inhibit the β-glucuronidase enzyme, while none inhibited the α-chymotrypsin enzyme.     

Steroid-1-dehydrogenase of <Emphasis Type="Italic">Mycobacterium</Emphasis> sp. VKM Ac-1817D strain producing 9α-hydroxy-androst-4-ene-3,17-dione from sitosterol

The strain of Mycobacterium sp. VKM Ac-1817D forms 9α-hydroxy-androst-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol. The formation of 9-OH-AD was accompanied with its partial destruction due to residual steroid-1-dehydrogenase (St1DH) activity. The activity was found to be induced by androst-4-ene-3,17-dione (AD), while other intermediates of sitosterol oxidation did not influence 1(2)-dehydrogenation. The enzyme is located mainly in the cytosolic fraction. The cytosolic St1DH (dimer, M _r∼58 kDa) was partially purified by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose and Phenyl-Sepharose, and gel filtration on Bio-Gel A-0.5M. It expressed the St1DH activity toward both AD and 9-OH-AD.     

Metabolism of 17-hydroxyprogesterone by a Bacillus species.

Fermentation of 17-hydroxyprogesterone with a Bacillus species (IICB-301) in a modified nutrient medium under aerobic conditions yielded androst-4-ene-3,17-dione and 15 alpha,17-dihydroxypregn-4-ene-3,20-dione in addition to a new pregnane analogue, 6 beta,17,20 alpha-trihydroxypregn-4-ene-3-one. Each microbial metabolite was characterized by the application of various spectroscopic techniques. The availability of the new metabolite, 6 beta,17,20 alpha-trihydroxypregn-4-ene-3-one, enabled complete elucidation of its 13C-n.m.r. spectrum.     

Steroidal Metabolites Transformed by <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> Cultures Block Breast Cancer Estrogen Biosynthesis

Suspension of cultured cells of Marchantia polymorpha have the potential to hydrogenate the olefinic bonds present in androst-1,4-dien-3,17-dione (boldione, 1) to afford dihydroandrost-3,17-dione derivatives including: androst-4-ene-3,17-dione (androstenedione, 4-AD, 2), 5α-androstane-3,17-dione (androstenedione, AD, 4), and the less abundant metabolite 5α-androst-1-ene-3,17-dione (1-androstenedione, 1-AD, 3). After isolation and purification, these metabolites were characterized on the basis of spectroscopic analyses using 1D and 2D NMR as well as mass spectrometry. Cytotoxicity of the biotransformation products against breast adenocarcinoma cells (MCF-7) was assessed by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and cell death (apoptosis or necrosis) was assayed by acridine orange/ethidium bromide staining. Aromatase (cytochrome P450 19 enzyme, CYP19) inhibitory activity was measured by a tritiated water release assay and by direct measurement of bio-transformed steroids using the tritium labeled substrate ³H-androst-4-ene-3,17-dione. CYP19 mRNA expression in MCF-7 cells was analyzed by real-time PCR. Steroidal products 3 and 4 revealed a highly significant inhibition of MCF-7 cell growth that was predominantly due to apoptosis not necrosis. Steroidal products 3 and 4 are both potent inhibitors of aromatase activity and CYP19 mRNA expression, while 2 is a known substrate for aromatase. These data establish that metabolites 3 and 4 are potent chemical agents against breast cancer via aromatase inhibitory mechanism. Results were interpreted via virtual docking of the biotransformation products to the human placental aromatase active site.     

Biotransformation of dehydroepiandrosterone with Macrophomina phaseolina and β-glucuronidase inhibitory activity of transformed products

The biotransformation of dehydroepiandrosterone (1) with Macrophomina phaseolina was investigated. A total of eight metabolites were obtained which were characterized as androstane-3,17-dione (2), androst-4-ene-3,17-dione (3), androst-4-ene-17β-ol-3-one (4), androst-4,6-diene-17β-ol-3-one (5), androst-5-ene-3β,17β-diol (6), androst-4-ene-3β-ol-6,17-dione (7), androst-4-ene-3β,7β,17β-triol (8), and androst-5-ene-3β,7α,17β-triol (9). All the transformed products were screened for enzyme inhibition, among which four were found to inhibit the β-glucuronidase enzyme, while none inhibited the α-chymotrypsin enzyme.     

HPLC analysis of PAMAM dendrimer based multifunctional devices

6-OXO, a new nutritional supplement commercially available on the internet, is sold as an aromatase-inhibitor and contains androst-4-ene-3,6,17-trione as active ingredient. This anabolic steroid is a prohibited substance in sports. Androst-4-ene-3,6,17-trione is metabolised to androst-4-ene-6alpha-ol-3,17-dione and androst-4-ene-6alpha,17beta-diol-3-one. A fast, sensitive and accurate LC/MS method was developed and validated for the quantification of androst-4-ene-3,6,17-trione and its metabolites in urine. The method is capable of determining the stereochemical position of the hydroxy-group at C-6 of the metabolites and consists of a liquid-liquid extraction step with diethylether after enzymatic hydrolysis, followed by separation on a reversed phase column. Ionisation of the analytes is carried out using atmospheric pressure chemical ionisation. The limit of quantification of the method was 5 ng/mL for all compounds. The accuracy ranged from 14.8 to 1.3% for androst-4-ene-3,6,17-trione, 9.4 to 1.6% for androst-4-ene-6alpha-ol-3,17-dione and 4.1 to 3.2% for androst-4-ene-6alpha,17beta-diol-3-one in the range of 5-1000 ng/mL. Using this method androst-4-ene-6alpha-ol-3,17-dione was identified as a major urinary metabolite, whereas androst-4-ene-6alpha,17beta-diol-3-one as a minor metabolite. While the parent compound is predominantly excreted in conjugated form, both metabolites are solely excreted as conjugates.     

Studies on an extract of rat testicular microsomal fraction that catalyses the transformation of progesterone into 17-hydroxyprogesterone and androgens

The supernatant obtained by centrifugation of Triton N-101-treated freeze-dried rat testicular microsomal fraction at 105000g(av.) for 2h transformed progesterone into testosterone via 17-hydroxypregn-4-ene-3,20-dione and androst-4-ene-3,17-dione. Hydroxylation at C-17 of 3beta-hydroxypregn-5-en-20-one and deoxycorticosterone was not observed. Non-haem iron protein, cytochrome P-450 and material with NADPH dehydrogenase activity were precipitated by 40% saturation of the supernatant with ammonium sulphate; however, it was not possible to establish the participation of these substances in the 17alpha-hydroxylase and side-chain-cleavage activities also present in the precipitate. The results of gel-filtration chromatography indicated that the Triton N-101 extract consisted primarily of a suspension of small particles of microsomes and that the progesterone 17-hydroxylase and the 17-hydroxypregn-4-ene-3,20-dione side-chain-cleavage enzyme were not in true solution.     

Subcellular localization and properties of mouse adrenal C19-steroid 5beta-reductase.

The localization and some characteristics of mouse adrenal C19-steroid 5 beta-reductase were determined by the incubation of subcellular fractions of mouse adrenal tissue with [7 alpha-3H]androst-4-ene-3,17-dione. This enzyme was present only in the soluble fraction and was NADPH-dependent, although a small activity in the presence of NADH was also detected. The soluble fraction also contained 3alpha-, 3beta- and a small amount of 17 beta-hydroxy steroid dehydrogenase. These and other steroid-metabolizing enzymes present in the remaining subcelluar fractions are also described briefly. To measure 5 beta-androstane-3,17-dione production by the mouse adrenal soluble fraction, all 5 beta products first had to be oxidized to 5 beta-androstane-3,17-dione, and the recovery of radio-activity between the substrate androst-4-ene-3,17-dione and product 5 beta-androstane-3,17-dione of 96.1 +/-3.2% validated this technique. C19-steroid 5 beta-reductase has a pH optimum of 6.5 and at low substrate concentrations the Km and Vmax. for 5 beta reduction of [7 alpha-3H]androst-4-ene-ene-3,17-dione was 2.22 times 10(-6) "/- 0.48 times 10(-6) M and 450+/- 53 pmol/min per mg of protein respectively. At high substrate concentration, inhibition of the reaction occurred, which was shown to be due to increasing product concentration.     

Microbial transformation of androst-4-ene-3,17-dione by Beauveria bassiana

The microbial transformation of androst-4-ene-3,17-dione (I) by the fungus Beauveria bassiana CCTCC AF206001 has been investigated using pH 6.0 and 7.0 media. Two hydroxylated metabolites were obtained with the pH 6.0 medium. The major product was 11alpha-hydroxyandrost-4-ene-3,17-dione (II) whereas the minor product was 6beta,11alpha-dihydroxyandrost-4-ene-3,17-dione (III). On the other hand, four hydroxylated and/or reduced metabolites were obtained with the pH 7.0 medium. The major product was 11alpha,17beta-dihydroxyandrost-ene-3-one (V) and the minor products were 17beta-hydroxyandrost-ene-3-one (IV), 6beta,11alpha,17beta-trihydroxyandrost-ene-3-one (VI) and 3alpha,11alpha,17beta-trihydroxy-5alpha-androstane (VII). The products were purified by chromatographic methods, and were identified on the basis of spectroscopic methods. This fungus strain is clearly an efficient biocatalyst for 11alpha-hydroxylation and reduction of the 17-carbonyl group.     

Biotransformation of 3b-Hydroxyandrost-5-en-17-one by Cell Suspension Cultures of Catharanthus roseus

Catharanthus roseus (L.) G. Don cell suspension cultures were used to transform 3b-hydroxyandrost-5-en-17-one, the products were isolated by chromatographic methods. Their structures were established by means of NMR and MS spectral analyses. Nine metabolites were respectively elucidated as: androst-4-ene-3,17-dione (Ⅰ), 6a-hydroxyandrost-4-ene-3,17-dione (Ⅱ), 6a,17b-dihydroxyandrost-4-en-3-one (Ⅲ), 6b-hydroxyandrost-4-ene-3,17-dione (Ⅳ), 17b-hydroxyandrost-4-en-3-one (Ⅴ), 15a,17b-dihydroxyandrost-4-en-3-one (Ⅵ), 15b,17b-dihydroxyandrost-4-en-3-one (Ⅶ), 14a-hydroxyandrost-4-ene-3,17-dione (Ⅷ), 17b-hydroxyandrost-4-ene-3,16-dione (Ⅸ). It is the first time to obtain the above compounds by biotransformation with Catharanthus roseus cell cultures.     

Significance of the steroid-induced type I spectral change in steroid C-21 hydroxylase system of bovine adrenocortical microsomes

The hydroxylyzable steroid 17-hydroxyprogesterone as well as the nonhydroxylyzable steroid androst-4-ene-3,17-dione induce Type I spectral change in cytochrome P-450, the oxygen activating component of the C-21 hydroxylase system. The data presented show quantitative relationship between the Type I spectral change and (1) the steroid-dependent NADPH oxidation; (2) the steroid-dependent increase in the steady-state level of P-450·CO and (3) the rate of C-21 hydroxylation in the case of 17-hydroxyprogesterone. The results indicate that the Type I spectral change is a reflection of the amount of the cytochrome activated for redox reactions and is independent of steroid hydroxylation.     

Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria <Emphasis Type="Italic">Mycobacterium neoaurum,Pimelobacter simplex</Emphasis>, and <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5–15 μm) or the transformation in the presence of randomly methylated β-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The crude product ADD was obtained in 75% yield, based on phytosterol. It contained as by-products 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment—the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD—no by-products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5% AD was obtained. The yield of 9-OH-AD (m.p. 218–220°C) based on transformed phytosterol was 56%.     

Microbial transformation of 3-hydroxy-5,6-cyclopropanocholestanes--an alternative route to 6-methylsteroids

Incubation of 3beta-hydroxy-5,6alpha-cyclopropano-5alpha-cholestane (4), 3beta-hydroxy-5,6beta-cyclopropano-5beta-cholestane (5), and 3beta-hydroxy-5,6alpha-cyclopropano-5alpha-cholest-7-e ne (6) with Mycobacterium sp. (NRRL B-3805) gave a mixture of side chain cleaved 17-keto steroids as the major products in 52, 57, and 69% yields, respectively. Among these 17-keto steroids, the cyclopropyl ring eliminated product, androst-4-ene-3,17-dione (9), was isolated in 6, 4, and 8% yields, respectively. A cyclopropyl ring migration product, 6alpha,7alpha-cyclopropanoandrost-4-ene-3,17-dione (16), was isolated from the incubation mixture of 6 in 4% yield, also 10% yield of 16 was obtained when 5, 6alpha-cyclopropano-5alpha-androst-7-ene-3,17-dione (12) was incubated. The cyclopropyl ring opening and subsequent reduction followed by oxidation of the two major biotransformation products, 5, 6beta-cyclopropano-5beta-androsta-3,17-dione (10) and 5, 6alpha-cyclopropano-5alpha-androsta-3,17-dione (7), gave 6beta- and 6alpha-methylandrost-4-ene-3,17-dione in 60, and 45% yields, respectively.     

Hormonal steroids in the tissue of induced rat mammary tumours

The possible presence of steroids in the tissue of induced hormone-dependent rat mammary tumours was investigated. The method used involves a preliminary extraction of tumours followed by chemical separation and thin-layer chromatography. The identified compounds were cholesterol, androst-4-ene-3,17-dione, 5β-androst-1-ene-3,17-dione, androsta-1,4-diene-3,17-dione and oestrone. This is the first report of the presence of these steroids in the tissue of an experimental tumour of a non-endocrine organ. In particular 5β-androst-1-ene-3,17-dione has not previously been identified from natural sources.     

Studies on the microbial transformation of androst-1,4-dien-3,17-dione with Acremonium strictum

The strain of Acremonium strictum PTCC 5282 was applied to investigate the biotransformation of androst-1,4-dien-3,17-dione (I; ADD). Microbial products obtained were purified by preparative TLC and the pure metabolites were characterized on the basis of their spectroscopic features (¹³C NMR, ¹H NMR, FTIR, MS) and physical constants (melting points and optical rotations). The 15α-Hydroxyandrost-1,4-dien-3,17-dione (II), 17β-hydroxyandrost-1,4-dien-3-one (III), androst-4-en-3,17-dione (IV; AD), 15α-hydroxyandrost-4-en-3,17-dione (V), 15α,17β-dihydroxyandrost-1,4-dien-3-one (VI) and testosterone (VII) were produced during this fermentation. Formation of the 15α,17β-dihydroxy derivative of ADD is reported for the first time during steroid biotransformation. The bioconversion reactions observed were 1,2-hydrogenation, 15α-hydroxylation and 17-ketone reduction. From the time course profile of this biotransformation, ketone reduction and 1,2-hydrogenation were observed from the first day of fermentation while 15α-hydroxylation occurred from the third day. Optimum concentration of the substrate, which gave the maximum bioconversion efficiency, was 0.5 mg ml⁻¹ in one batch. The highest yield of the microbial products recorded in this work was achieved within the pH range 6.5–7.3 and at the temperature of 27 °C.