Metabolism of 19-methyl substituted steroids and a proposal for the third aromatase monooxygenation

The article summarizes the results of recent studies on the metabolism of 10-ethylestr-4-ene-3,17-dione, 10-[(1R)-1-hydroxyethyl]-,and 10-[(1S)-1-hydroxyethyl]estr-4-ene-3, 17-dione, in placenta. These compounds are the 19-methyl analogs of androstenedione, 19-hydroxyandrostenedione, and 19-oxoandrostenedione, respectively. No conversion of 10-ethylestr-4-ene-3,17-dione to either estrogens or oxygenated metabolites was detected. Both 10-[(1R)-1-hydroxyethyl]- and 10-[(1S)-1-hydroxyethyl]estr-4-ene-3, 17-dione were oxygenated to 10-(1,1-dihydroxyethyl)estr-4-ene-3,17-dione and isolated following in situ dehydration as 10-acetylestr-4-ene-3,17-dione. Evidence for the involvement of aromatase in these conversions is discussed. No conversion of 10-acetylestr-4-ene-3,17-dione to either estrogens or other oxygenated products was detected. These results lead us to propose a new mechanism for the third aromatase monooxygenation. We propose that the third oxygenation is initiated by 1β-hydrogen abstraction at C₁ of 19,19-dihydroxyandrostenedione, followed by homolytic cleavage of the C₁₀−C₁₉ bond with concurrent formation of a Δ^1(10),4−3-ketosteroid and a C₁₉ carbon radical, and terminated by oxygen rebound at C₁₉.     

Therapy of Cushing''s syndrome with steroid biosynthesis inhibitors

Several substances with different inhibitory effects on adrenal steroid biosynthesis were investigated in patients with Cushing's syndrome. It has been shown that trilostane, a 3β-hydroxysteroid-dehydrogenase inhibitor, is not potent enough to block cortisol biosynthesis in patients with hypercortisolism. Aminoglutethimide inhibits side chain cleavage of cortisol synthesis, but it has been demonstrated that the blocking effect on cortisol secretion is not strong enough to normalize urinary cortisol excretion in patients with Cushing's disease. For metyrapone, an inhibitor of adrenal 11β-hydroxylase, promising results were reported for the treatment of Cushing's syndrome. However, the drug has several side effects and depending on the definition of the desired reduction of cortisol secretion a true remission was only found in a minority of patients. The antifungal drug ketoconazole in vitro predominantly blocks 17,20-desmolase (IC₅₀ 1 μM) and to a lesser extent 17-hydroxylase (IC₅₀ 10 μM) and 11β-hydroxylase (IC₅₀ 15–40 μM). Therefore, ketoconazole in vivo most potently suppresses androgen secretion and only to a lesser extent cortisol biosynthesis. Several therapeutic trials with ketoconazole treatment in patients with pituitary Cushing's disease showed various remission rates between 30 and 90%. In contrast, in almost all patients with benign, primary adrenal Cushing's syndrome cortisol levels were normalized. In patients with ectopic ACTH syndrome ketoconazole was effective in about 50% of all reported cases, while cortisol hypersecretion due to adrenocortical carcinoma was only rarely inhibited by ketoconazole. The main side effect of ketoconazole treatment was liver toxicity which occurred in 12% of all treated patients. In contrast to ketoconazole, the narcotic drug etomidate shows a strong inhibitory effect on 11β-hydroxylase (IC₅₀ 0.03–0.15 μM) but only a weak inhibition of 17,20 desmolase (IC₅₀ 380 μM). This correlates with in vivo studies where even low, non-hypnotic doses of etomidate induced a pronounced fall in serum cortisol levels in normals and in patients with Cushing's syndrome. However, its clinical use is limited by its mandatory intravenous application and its sedative effects. In conclusion, ketoconazole remains the only available steroid-inhibitory drug for a therapeutic trial in patients with Cushing's syndrome who cannot be treated definitively by surgery.     

Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera:Dytiscidae): I. Metabolism of androst-4-en-3,17-dione (AD)

Two Bacillus strains were isolated from the foregut of the water beetle Agabus affinis (Payk.) and tested for their steroid transforming ability. After incubation with androst-4-en-3,17-dione (AD), 13 different transformation products were detected. AD was hydroxylated at C₆, C₇, C₁₁ and C₁₄, resulting in formation of 6β-, 7-, 11- and 14-hydroxy-AD. One strain also produced small amounts of 6β,14-dihydroxy-AD. Partly, the 6β-hydroxy group was further oxidized to the corresponding 6-oxo steroids. In addition, a specific reduction of the Δ⁴-double bond was observed, leading to the formation of 5-androstane derivatives. In minor yields the carbonyl functions at C₃ and C₁₇ were reduced leading to the formation of 3ξ-OH or 17β-OH steroids. EI mass spectra of the trimethylsilyl and O-methyloxime trimethylsilyl ether derivatives of some transformation products are presented for the first time.     

Endogenous inhibitors (GALFs) of 11beta-hydroxysteroid dehydrogenase isoforms 1 and 2: derivatives of adrenally produced corticosterone and cortisol

Two isoforms of 11β-HSD exist; 11β-HSD1 is bi-directional (the reductase usually being predominant) and 11β-HSD2 functions as a dehydrogenase, conferring kidney mineralocorticoid specificity. We have previously described endogenous substances in human urine, “glycyrrhetinic acid-like factors (GALFs)”, which like licorice, inhibit the bi-directional 11β-HSD1 enzyme as well as the dehydrogenase reaction of 11β-HSD2.

Many of the more potent GALFs are derived from two major families of adrenal steroids, corticosterone and cortisol. For example, 35-tetrahydro-corticosterone, its derivative, 35-tetrahydro-11β-hydroxy-progesterone (produced by 21-deoxygenation of corticosterone in intestinal flora); 35-tetrahydro-11β-hydroxy-testosterone (produced by side chain cleavage of cortisol); are potent inhibitors of 11β-HSD1 and 11β-HSD2-dehydrogenase, with IC₅₀'s in range 0.26–3.0 μM, whereas their 11-keto-35-tetrahydro-derivatives inhibit 11β-HSD1 reductase, with IC₅₀'s in range 0.7–0.8 μM (their 35β-derivatives being completely inactive).

Inhibitors of 11β-HSD2 increase local cortisol levels, permitting it to act as a mineralocorticoid in kidney. Inhibitors of 11β-HSD1 dehydrogenase/11β-HSD1 reductase serve to adjust the set point of local deactivation/reactivation of cortisol in vascular and other glucocorticoid target tissues, including adipose, vascular, adrenal tissue, and the eye. These adrenally derived 11-oxygenated C₂₁- and C₁₉-steroidal substances may serve as 11β-HSD1- or 11β-HSD2-GALFs. We conclude that adrenally derived products are likely regulators of local cortisol bioactivity in humans.     

Cortisol metabolism in vitro—III. Inhibition of microsomal 6β—hydroxylase and cytosolic 4-ene-reductase

The in vitro metabolism of cortisol in human liver fractions is highly complex and variable. Cytosolic metabolism proceeds predominantly via A-ring reduction (to give 3,5β-tetrahydrocortisol; 3,5β-THF), while microsomal incubations generate upto 7 metabolites, including 6β-hydroxycortisol (6β-OHF), and 6β-hydroxycortisone (6β-OHE), products of the cytochrome P450 (CYP) 3A subfamily. The aim of the present study was, therefore, to examine two of the main enzymes involved in cortisol metabolism, namely, microsomal 6β-hydroxylase and cytosolic 4-ene-reductase. In particular, we wished to assess the substrate specificity of these enzymes and identify compounds with inhibitory potential. Incubations for 30 min containing [³H]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The K_m value for 6β-OHF and 6β-OHE formation was 15.2 ± 2.1 μM (mean ± SD; n = 4) and the V_max value 6.43 ± 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6β-hydroxylase was ketoconazole (K_i = 0.9 ± 0.4 μM; N = 4), followed by gestodene (K_i = 5.6 ± 0.6 μM) and cyclosporine (K_i = 6.8 ± 1.4 μM). Both betamethasone and dexamethasone produced some inhibition (K_i = 31.3 and 54.5 μ, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The K_m value for cortisol 4-ene-reductase was 26.5 ± 11.2 μM (n = 4) and the V_max value 107.7 ± 46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione (K_i = 17.8 ± 3.3 μM) and gestodene (K_i = 23.8 ± 3.8 μM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive.     

Kinetic analysis of enzymic activities: Prediction of multiple forms of 17β-hydroxysteroid dehydrogenase

An overview of the application of kinetic methods to the delineation of 17β-hydroxysteroid dehydrogenase (17β-HSD) heterogeneity in mammalian tissues is presented. Early studies of 17β-HSD activity in animal liver and kidney subcellular fractions were suggestive of multiple forms of the enzyme. Subsequently, detailed characterization of activity in cytosol and subcellular membrane fractions of human placenta, with particular emphasis on inhibition kinetics, yielded evidence of two kinetically-differing forms of 17β-HSD in that organ. Gene cloning and transfection experiments have confirmed the identity of these two proteins as products of separate genes. 17β-HSD type 1 is a cytosolic enzyme highly specific for C₁₈ steroids such as 17β-estradiol (E₂) and estrone (E₁). 17β-HSD type 2 is a membrane bound enzyme reactive with testosterone (T) and androstenedione (A), as well as E₂ and E₁. Useful parameters for the detection of multiple forms of 17β-HSD appear to be the E₂/T activity ratio, NAD/NADP activity ratios, steroid inhibitor specificity and inhibition patterns over a wide range of putative inhibitor concentrations. Evaluation of these parameters for microsomes from samples of human breast tissue suggests the presence of 17β-HSD type 2. The 17β-HSD enzymology of human testis microsomes appears to differ from placenta. Analysis of human ovary indicates granulosa cells are particularly enriched in the type 1 enzyme with type 2-like activity in stroma/theca. Mouse ovary appears to contain forms of 17β-HSD which differ from 17β-HSD type 1 and type 2 in their kinetic properties.     

Action of endogenous steroid inhibitors of brain aromatase relative to fadrozole

To study mechanisms of aromatase inhibition in brain cells, a highly effective non-steroidal aromatase inhibitor (Fadrozole; 4-[5,6,7,8-tetra-hydroimidazo-(1,5-a)-pyridin-5-yl] benzonitrile HCl; CGS 16949A) was compared with endogenous C-19 steroids, known to be formed in the preoptic area, which inhibit oestrogen formation. Using a sensitive in vitro tritiated water assay for aromatase activity in avian (dove) preoptic tissue, the order of potency, with testosterone as substrate was: Fadrozole (K_i < 1 × 10⁻⁹ M) > 4-androstenedione 5-androstanedione > 5-dihydrotestosterone (K_i = 6 × 10⁻⁸ M) > 5β-androstanedione > 5β-dihydrotestosterone (K_i = 3.5 × 10⁻⁷ M) > 5-androstane-3, 17β-diol (K_i = 5 × 10⁻⁶ M) > 5β-androstane-3β,17β-diol. Five other steroids, 5β-androstane-3,17β-diol, 5-androstane-3β,17β-diol, progesterone, oestradiol and oestrone, showed no inhibition at 10⁻⁴ M. The kinetics indicate that endogenous C-19 steroids show similar competitive inhibition of the aromatase as Fadrozole. Mouse (BALB/c) preoptic aromatase was also inhibited by Fadrozole. We conclude that endogenous C-19 metabolites of testosterone are effective inhibitors of the brain aromatase, and suggest that they bind competitively at the same active site as Fadrozole.     

Kinetic analysis of duodenal and testicular cytochrome P450c17 in the rat

In the adult rat, the duodenal tissue of both sexes can convert progesterone to 17-hydroxyprogesterone, androstenedione and testosterone. The transition from C₂₁ to C₁₉ steroids is apparently controlled by the same cytochrome P450c17 expressed in the testis, which catalyzes both 17-hydroxylation and C-17,20 bond scission at a single bifunctional active site. The kinetic parameters of this enzyme were measured at the steady state for both reactions using [1,2-³H]progesterone and [1,2-³H]17-hydroxyprogesterone as substrates. In the testis and male and female duodena, the K_m values for progesterone 17-hydroxylation were 14.2, 23.8 and 23.2 nM, whereas the V_max values were 105, 3.5 and 3.1 pmol/mg protein/min, respectively. With respect to C-17,20 lyase activity, the K_m values for exogenous 17-hydroxyprogesterone were 525, 675 and 637 nM, whereas the V_max values were 283, 7.8 and 7.8 pmol/mg protein/min, respectively. However, when the K_m values were calculated with respect to intermediate 17-hydroxyprogesterone formed from progesterone, they were similar to the K_m values for 17-hydroxylase, being 15, 31.4 and 24.8 nM, whereas the V_max values were 26.3, 2 and 1.8 pmol/mg protein/min, respectively. The similarity of K_m values is due to the fact that the relative androgen formation efficiency (bond scission events/total 17-hydroxylation events ratio) was remarkably constant in both testicular and duodenal incubates, irrespective of progesterone concentration. Efficiency values were 2-fold higher in duodenal tissue (0.54) than in testis (0.25). Estradiol-17β inhibited 17-hydroxylation but not bond scission on intermediate 17-hydroxyprogesterone, because it did not affect the efficiency value. Rat duodenal P450c17 has the same substrate affinity, a lower specific activity and a higher androgen formation efficiency than testicular P450c17.     

Bovine adrenal cytochrome P-450(11 beta)-mediated conversion of 11-deoxycortisol to 18- and 19-hydroxy derivatives; structural analysis by 1H-NMR.

Incubation of 11-deoxycortisol with a cytochrome P-450(11β)-reconstituted system yielded, in addition to cortisol, several new steroid products. In this study, the structures of the three steroid products were elucidated. Retention time of the first product (Peak 2 substance) coincided with that of authentic 18-hydrocortisol on reverse phase HPLC. To further confirm the chemical identity of this product, the purified sample was subjected to ¹H-NMR analysis. The spectrum was essentially identical to that of 18-hydrocortisol. The retention time of the second product (Peak 3 substance) did not coincide with those of commonly occuring steroids. The one- and two-dimension ¹H-NMR spectra provided strong evidence for its structure of 19-hydroxy-11-deoxycortisol. The retention time of the third product (Peak 4 substance) did not coincide with those of commonly occuring steroids. The ¹H-NMR spectrum showed the presence of signals of 19-CH₃ and 18-CH₂ protons. There was also evidence that this product is not hydroxylated at the 11-position. Further analysis of the COSY spectra identified its structure as 18-hydroxy-11-depxycortisol. From these results, we conclude that bovine P-450(11β)deoxycortisol can catalyze the hydroxylation of 11-deoxycortisol at 11β-18- and 19-positions and produce cortisol, 18-hydroxy-11-deoxycortisol, 18-hydroxycortisol and 19-hydroxy-11-deoxycortisol.     

Extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by a benign testicular leydig cell tumor

We present an unusual case with bilateral testicular Leydig cell tumors displaying extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase. Histological examination of a 38-yr-old man infertile due to azoospermia showed him to have bilateral testicular Leydig cell tumors. The in vitro steroidogenic potential of the tumors and their adjacent testicular tissue was evaluated using organ culture. Tumor tissue was found to secrete deoxycorticosterone (DOC), corticosterone (B) and cortisol, which are not produced in normal adult testis, into the medium, while testicular tissue adjacent to the tumors secreted a small amount of DOC and B. Northern blot analysis with cytochrome P-450_C21 complementary DNA (cDNA) and P-450_11β cDNA as probes revealed that the tumor contained a considerable amount of mRNA for P-450_C21 and P-450_11β, while the mRNAs were not detected in the testicular tissues adjacent to the tumors. It is suggested that the high local levels of estrogen and/or progesterone within the Leydig cell tumors and their adjacent testicular tissues induced extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by the tumors and their adjacent testicular tissues.     

Regulation of the CYP27B1 5'-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.     

Investigations into the biosynthetic pathways for classical and ring B-unsaturated oestrogens in equine placental preparations and allantochorionic tissues

In on-going studies of ‘classical’ and ring B-unsaturated oestrogens in equine pregnancy, the products of metabolism of [2,2,4,6,6-²H₅]-testosterone and [16,16,17-²H₃]-5,7-androstadiene-3β,17β-diol with equine placental subcellular preparations and allantochorionic villi have been identified. Using mixtures of unlabelled and [²H]-labelled steroid substrates has allowed the unequivocal identification of metabolites by twin-ion monitoring in gas chromatography–mass spectrometry (GC–MS). Two types of incubation were used: (i) static in vitro and (ii) dynamic in vitro. The latter involved the use of the Oxycell™ cartridge (Integra Bioscience Systems, St Albans, UK) whereby the tissue preparation was continuously supplied with supporting medium plus appropriate cofactors in the presence of uniform oxygenation. [²H₅]-Testosterone was converted into [²H₄]-oestradiol-17β, [²H₄]-oestrone and [²H₃]-6-dehydro-oestradiol-17 in both placental and chorionic villi preparations, but to a greater extent in the latter, confirming the importance of the chorionic villi in oestrogen production in the horse.

On the basis of GC–MS characteristics (M⁺ m/z 477/482 (as O-methyl oxime-trimethyl silyl ether), evidence for 19-hydroxylation of testosterone was found in static incubations, while the presence of a 6-hydroxy-oestradiol-17 was recorded in dynamic incubations (twin peaks in the mass spectrum at m/z 504/507, the molecular ion M⁺). It was not possible to determine the configuration at C-6. The formation of small, but significant, quantities of [²H₄]-17β-dihydroequilin was also shown, and a biosynthetic pathway is proposed.

In static incubations of placental microsomal fractions, the 17β-dihydro forms of both equilin and equilenin were shown to be major metabolites of [²H₃]-5,7-androstadiene-3,17-diol. Using static incubations of chorionic villi, the deuterated substrate was converted into the 17β-dihydro forms of both equilin and equilenin, together with an unidentified metabolite (base peak, m/z 504/506). The isomeric 17-dihydroequilins were also obtained using the dynamic in vitro incubation of equine chorionic villi, together with the 17β-isomer of dihydroequilenin. Confirmation of the identity of 17β-dihydroequilin and 17β-dihydroequilenin was obtained by co-injection of the authentic unlabelled steroids with the phenolic fraction obtained from various incubations. Increases in the peak areas for the non-deuterated steroids (ions at m/z 414 (17β-dihydroequilin) and 412 (17β-dihydroequilenin) (both as bis-trimethyl silyl ether derivatives) were observed. Biosynthetic pathways for formation of the ring B-unsaturated oestrogens from 5,7-androstadiene-3β,17β-diol are proposed.     

The molecular biology of androgenic 17β-hydroxysteroid dehydrogenases

The enzyme 17β-hydroxysteroid dehydrogenase (17β-HSD) catalyzes the 17β-oxidation/reduction of C₁₈- and C₁₉-steroids in a variety of tissues. Three human genes encoding isozymes of 17β-HSD, designated 17β-HSD types 1, 2 and 3 have been cloned. 17β-HSD type 1 (also referred to as estradiol 17β-dehydrogenase) catalyzes the conversion of estrone to estradiol, primarily in the ovary and placenta. The 17β-HSD type 2 is expressed to high levels in the liver, secretory endometrium and placenta. The type 2 isozyme catalyzes the oxidation of androgens and estrogens equally efficiently. Also, the enzyme possesses 20-HSD activity demonstrated by its ability to convert 20-dihydro-progesterone to progesterone. Testicular 17β-HSD type 3 catalyzes the conversion of androstenedione to testosterone, dehydroepiandrosterone to 5-androstenediol and estrone to estradiol. The 17β-HSD3 gene is mutated in male pseudohermaphrodites with the genetic disease 17β-HSD deficiency.     

Molecular biology of 11β-hydroxylase and 11β-hydroxysteroid dehydrogenase enzymes

There are two steroid 11β-hydroxylase isozymes encoded by the CYP11B1 and CYP11B2 genes on human chromosome 8q. The first is expressed at high levels in the normal adrenal gland, has 11β-hydroxylase activity and is regulated by ACTH. Mutations in the corresponding gene cause congenital adrenal hyperplasia due to 11β-hydroxylase deficiency; thus, this isozyme is required for cortisol biosynthesis. The second isozyme is expressed at low levels in the normal adrenal gland but at higher levels in aldosterone-secreting tumors, and has 11β-hydroxylase, 18-hydroxylase and 18-oxidase activities. The corresponding gene is regulated by angiotensin II, and mutations in this gene are found in persons who are unable to synthesize aldosterone due to corticosterone methyloxidase II deficiency. Thus, this isozyme is required for aldosterone biosynthesis.

Cortisol and aldosterone are both effective ligands of the “mineralocorticoid” receptor in vitro, but only aldosterone is a potent mineralocorticoid in vivo. This apparent specificity occurs because 11β-hydroxysteroid dehydrogenase in the kidney converts cortisol to cortisone, which is not a ligand for the receptor. This enzyme is a “short-chain” dehydrogenase which is encoded by a single gene on human chromosome 1. It is possible that mutations in this gene cause a form of childhood hypertension called apparent mineralocorticoid excess, in which the mineralocorticoid receptor is not protected from high concentrations of cortisol.     

Some observations on the measurement of estradiol-17β in human plasma by radioimmunoassay

The use of specific and non-specific antisera for estradiol-17β (E₂17β) were compared in the radioimmunoassay of the steroid. The effects of various “blank” mateirials on the standard curve and on the accuracy of recovery of E₂17β added to plasma before and after chromatography on LH-20 Sephadex were examined. It was concluded that the use of the specific antiserum (anti-6-oxoE₂17β -6-(O-carboxymethyl)oxime-bovine serum albumin(antiE₂17β-6-BSA) was an improvement on the non-specific serum anti-E₂17β-17-hemisuccinyl-bovine serum albumin (antiE₂ 17β-17-BSA) following chromatography of extracts. However, although a precise result could be obtained with the anti-E₂17β-6-BSA without the Chromatographic step, recovery of E₂17β added to plasma was only possible if the step was included.

The cross-reactivity of estrone (E₁)with E₂17β using anti-E₂17β-17-BSA as defined by Abraham (J. Clin. Endocr. , 866 (1969) was examined under conditions of constant and of changing E₁:E₂17β ratio.     

细胞色素P450还原酶与CYP17的共表达及其功能分析*

17α-羟基黄体酮(17α-OH-PROG)是甾体激素类药物的关键中间体,其生物合成主要由细胞色素单加氧酶(CYP17)催化生成。在此过程中,细胞色素 P450还原酶(cytochrome P450 reductase,CPR)作为细胞色素P450 酶电子传递链的重要组成部分,直接影响CYP17的催化效率。为研究不同来源CPR与17α-羟化酶的适配性,首先以人源17α-羟化酶作为研究对象,构建了表达质粒pPIC3.5k-hCYP17,获得了重组毕赤酵母菌株。其次筛选获得3种不同来源CPR,构建了表达质粒 pPICZX-CPR,获得17α-羟化酶与CPR共表达菌株,并在毕赤酵母中进行转化实验,对转化产物进行薄层色谱(TLC)和高效液相色谱(HPLC)分析。结果显示,重组菌株具有17α-羟化酶活性,能够催化黄体酮生成目标产物17α-OH-PROG 以及副产物16α-羟基黄体酮(16α-OH-PROG)。不同来源的CPR与17α-羟化酶共表达与仅表达17α-羟化酶的产率相比均有所提高,其中hCPR-CYP17共表达菌株表现出最高的转化水平,17α-OH-PROG产率提高42%。上述结果表明:17α-羟化酶基因与CPR共表达能够提高其黄体酮17α-羟基化水平。为甾体黄体酮17α-羟基化的生物催化研究提供思路,对甾体药物的工业生产具有重要意义。     

Steroid production in toads

In Bufo arenarum, androgen biosynthesis occurs through a complete 5-ene pathway, including 5-androstane-3β,17β-diol as the immediate precursor of testosterone. Besides, steroidogenesis changes during the breeding period, turning from androgens to C₂₁-steroids such as 5-pregnan-3,20-diol, 3-hydroxy-5-pregnan-20-one and 5-pregnan-3,20-dione. In B. arenarum, steroid hormones are not involved in hCG-induced spermiation, suggesting that the steroidogenic shift to C₂₁-steroids during the breeding be not related to spermiation. The activity of 17-hydroxylase-C_17–20 lyase (CypP450_c17) decreases during the reproductive season, suggesting that this enzyme would represent a key enzyme in the regulation of seasonal changes. However, the increase in the affinity for pregnenolone of 3β-hydroxysteroid dehydrogenase (3HSD)/isomerase could also be involved. Moreover, the reduction in CypP450_c17 leading to a reduction in C₁₉-steroids, among them dehydroepiandrosterone (DHE), would contribute to the conversion of pregnenolone into progesterone, avoiding the non-competitive inhibition exerted by DHE on this transformation. Additionally, CypP450_c17 possesses a higher affinity for pregnenolone than for progesterone, explaining the predominance of the 5-ene pathway for testosterone biosynthesis. Animals in reproductive condition showed a significant reduction in circulating androgens, enhancing the physiological relevance of all the in vitro results. The in vitro effects of mGnRH and hrFSH on testicular steroidogenesis revealed that both hormones inhibited CypP450_c17 activity. In summary, these results demonstrate that, in B. arenarum, the change in testicular steroidogenesis during the reproductive period could be partially due to an FSH and GnRH-induced decrease in CypP450_c17 activity.     

Ionic iridium-gold and rhodium-gold perfluorobenzenethiolato binuclear complexes: syntheses and solution behavior

By reacting [(C₅Me₅)M(SR_F)₂] (forM = Ir, Rf = C₆F₅ (1a) or C₆F₄H-p (1b); for M = Rh, Rf = C₆F₅ (2a) or C₆F₄H-p (2a)) in toluene with Na[AuCl₄], ionic binuclear compounds with the general formula [(C₅Me₅)M(μ-SR_F)₂AuCl₂]Cl for M = Ir, R = C₆F₅ (3a) or C₆F₄H-p (3a); for M = Rh, R_F = C₆F₅ (4a) or C₆F₄H-p (4b) can be obtained, together with small amounts of [(C₅Me₅)₂Rh₂(μ-SR_F)(μ-Cl)₂]Cl (R_F = C₆F₅ (5a) or C₆F₄H-p (5b)) as by-products when 2a and 2b were used.     

Dialkyl-μ-ethylidene-μ-methylene-bis(pentamethylcyclopentadienyl)-dirhodium complexes: synthesis, spectra and decomposition reactions

The dialkyl-μ-ethylidene-μ-methylene-bis (pentamethylcyclopentadienyl)-dirhodium complexes [{(C₅Me₅)Rh}₂(μ-CH₂)(μ-CHMe) (R)₂] (4, P=Me; 5, Et; 6, n-Bu; 7, CH=CH₂; and 8, Z-CH=CHMe) have been prepared from RMgBr and [{(C₅Me₅)Rh}₂(μ-CH₂)(μ-CHMe)(X)₂] (2, X=Cl; 3, X=Br). Structures deduced from the NMR spectra show that the dialkyl complexes can exist in one trans and two cis forms. The decomposition of the dimethyl complex 4 is compared with that of the related di-μ-methylene complex; it reacts readily (30°C, MeCN solution) in the presence of one-electron oxidisers to give propene and methane and a little ethene and some butenes. Mass-spectrometric analysis of the ¹³C labelling in the organics originating from [{(C₅Me₅)Rh}₂(μ-CH₂)(μ-CHMe) (¹³CH₃)₂] shows that methane derives from the Rh---Me, ethene half from the ethylidene and half from coupling of Rh-methyl and a bridging methylene, while the propene arises almost entirely from the ethylidene and a rhodium methyl. The butenes come from coupling of ethylidene, methylene and a Rh-methyl, but only quite small amounts are formed; thus C+C coupling is the major decomposition path for the μ-ethylidenes, in contrast to the di-μ-methylene complexes where C+C+C coupling predominates. The divinyl complex [{(C₅Me₅)Rh}₂(μ-CH₂)(μ-CHMe) (CH=CH₂)₂] also underwent internal C+C coupling on reaction with AgBF₄ in MeCN to give a mixture of the allyl and methylallyl cations [(C₅Me₅)Rh(η³-CH₂CHCHR)(MeCN)]⁺(10, R=H; 11, R=Me).