Studies on the protective role of vitamin C and E against polychlorinated biphenyl (Aroclor 1254)—induced oxidative damage in Leydig cells

Free radical production and lipid peroxidation are potentially important mediators in testicular physiology and toxicology. Polychlorinated biphenyls (PCBs) are global environmental contaminants that cause disruption of the endocrine system in human and animals. The present study was conducted to elucidate the protective role of vitamin C and E against Aroclor 1254-induced changes in Leydig cell steroidogenesis and antioxidant system. Adult male rats were dosed for 30 days with daily intraperitoneal (ip) injection of 2 mg/kg Aroclor or vehicle (corn oil). One group of rats was treated with vitamin C (100 mg/kg bw/day) while the other group was treated with vitamin E (50 mg/kg bw/day) orally, simultaneously with Aroclor 1254 for 30 days. One day after the last treatment, animals were euthanized and blood was collected for the assay of serum hormones such as luteinizing hormone (LH), thyroid stimulating hormone (TSH), prolactin (PRL), triiodothyronine (T₃), thyroxine (T₄), testosterone and estradiol. Testes were quickly removed and Leydig cells were isolated in aseptic condition. Purity of Leydig cells was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining method. Purified Leydig cells were used for quantification of cell surface LH receptors and steroidogenic enzymes such as cytochrome P₄₅₀ side chain cleavage enzyme (P₄₅₀scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β- HSD). Leydig cellular enzymatic antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), γ-glutamyl transpeptidase (γ-GT), glutathione-S-transferase (GST) and non-enzymatic antioxidants such as vitamin C and E were assayed. Lipid peroxidation (LPO) and reactive oxygen species (ROS) were also estimated in Leydig cells. Aroclor 1254 treatment significantly reduced the serum LH, TSH, PRL, T₃, T₄, testosterone and estradiol. In addition to this, Leydig cell surface LH receptors, activities of the steroidogenic enzymes such as cytochrome P₄₅₀scc, 3β-HSD, 17β-HSD, antioxidant enzymes SOD, CAT, GPX, GR, γ-GT, GST and non-enzymatic antioxidants such as vitamin C and E were significantly diminished whereas, LPO and ROS were markedly elevated. However, the simultaneous administration of vitamin C and E in Aroclor 1254 exposed rats resulted a significant restoration of all the above-mentioned parameters to the control level. These observations suggest that vitamin C and E have ameliorative role against adverse effects of PCB on Leydig cell steroidogenesis.     

Increasing progesterone secretion and 3β-hydroxysteroid dehydrogenase activity of human cumulus cells and granulosa-lutein cells concurrent with successful fertilization of the corresponding oocyte

In many studies it has been documented that the induction of multiple follicular growth in humans results in an asynchrony between the degree of cumulus mucification, oocyte meiotic maturation, fertilizability, and follicular cell progesterone (P₄) secretion. The present study was carried out on oocytes enclosed in fully mucified cumulus. Thus, oocyte fertilizability was correlated to human cumulus cell (hCC) and human granulosa-lutein (G-L) cell competence for P₄ secretion in culture. In the G-L cells, P₄ secretion and percentage of cells manifesting 3β-hydroxysteroid dehydrogenase (3β-HSD) activity increased concurrently with the period of culture. In the hCC, however, P₄ secretion decreased concurrently with elongation of the culture period, whereas the percentage of 3β-HSD-positive cells increased. In hCC corresponding to the fertilized oocytes, P₄ accumulation in culture medium was 1.9-fold (P < 0.001) and 1.6-fold (P < 0.02) higher on days 0–3 and 3–5 of culture, respectively, as compared to P₄ accumulation in hCC of unfertilized oocytes. Also, in hCC corresponding to the fertilized oocytes, the degree of 3β-HSD activity was found to be significantly higher shortly after aspiration and after either 3 or 5 days, compared to hCC of unfertilized oocytes. In the G-L cells pooled from all follicles yielding mature cumulus-oocyte complexes, P₄ accumulation and percentage of 3β-HSD-positive cells increased concurrently with the increase in percentage of fertilized eggs of each individual woman. These results indicate that in stimulated cycles, follicles yielding mature cumulus-oocyte complex, oocyte fertilizability, and G-L cell or hCC competence for P₄ secretion are correlated and synchronous.     

Estradiol and progesterone synthesis in human placenta is stimulated by calcitriol

Calcitriol exerts a diverse range of biological actions including the control of growth and cell differentiation, modulation of hormone secretion, and regulation of reproductive function. The placenta synthesizes calcitriol through the expression of CYP27B1, but little is known about local actions of this hormone in the fetoplacental unit. The objective of this study was to investigate the effects of calcitriol upon progesterone (P₄) and estradiol (E₂) secretion in trophoblasts cultured from term human placenta. Cells were incubated in the presence of calcitriol for 18 h and pregnenolone or androstenedione were subsequently added as substrates for the 3β-hydroxysteroid dehydrogenase (3β-HSD) or P450-aromatase (CYP19), respectively. Calcitriol stimulated in a dose-dependant manner E₂ and P₄ secretion. The use of a selective inhibitor of PKA prevented the effects of calcitriol upon E₂ secretion, but not on P₄. These results show that calcitriol is a physiological regulator of placental E₂ and P₄ production and suggest a novel role for calcitriol upon placental steroidogenesis.     

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.     

Distribution of 17β-hydroxysteroid dehydrogenase gene expression and activity in rat and human tissues

The interconversion of estrone (E₁) and 17β-estradiol (E₂), androstenedione (4-ene-dione) and testosterone (T), as well as dehydroepiandrosterone and androst-5-ene-3β,17β-diol is catalyzed by 17β-hydroxysteroid dehydrogenase (17β-HSD). The enzyme 17β-HSD thus plays an essential role in the formation of all active androgens and estrogens in gonadal as well as extragonadal tissues. The present study investigates the tissue distribution of 17β-HSD activity in the male and female rat as well as in some human tissues and the distribution of 17β-HSD mRNA in some human tissues. Enzymatic activity was measured using ¹⁴C-labeled E₁, E₂, 4-ene-dione and T as substrates. Such enzymatic activity was demonstrated in all 17 rat tissues examined for both androgenic and estrogenic substrates. While the liver had the highestlevel of 17β-HSD activity, low but significant levels of E₂ as well as T formation were found in rat brain, heart, pancreas and thymus. The oxidative pathway (E₂→E₁, T→4-ene-dione) was favored over the reverse reaction in almost all rat tissues while in the human, almost equal rates were found in most of the 15 tissues examined. The widespread distribution of 17β-HSD in rat and human tissues clearly indicates the importance of this enzyme in peripheral sex steroid formation or intracrinology.     

Bovine adrenal 3beta-hydroxysteroid dehydrogenase (E.C. 1.1.1. 145)/5-ene-4-ene isomerase (E.C. 5.3.3.1): characterization and its inhibition by isoflavones

The isoflavones daidzein, genistein, biochanin A and formononetin inhibit potently and preferentially the γ-isozymes of mammalian alcohol dehydrogenase (γγ-ADH), the only ADH isozyme that catalyzes the oxidation of 3β-hydroxysteroids. Based on these results, we proposed that these isoflavones might also act on other enzymes involved in 3β-hydroxysteroid metabolism. Recently, we showed that they indeed are potent inhibitors of a bacterial β-hydroxysteroid dehydrogenase (β-HSD). To extend this finding to the mammalian systems, we hereby purified, characterized and studied the effects of isoflavones and structurally related compounds on, a bovine adrenal 3β-hydroxysteroid dehydrogenase (3β-HSD). This enzyme catalyzes the oxidation of 3β-hydroxysteroids but not 3-, 11β- or 17β-hydroxysteroids. The same enzyme also catalyzes 5-ene-4-ene isomerization, converting 5-pregnen 3, 20-dione to progesterone. The K_m values of its dehydrogenase activity determined for a list of 3β-hydroxysteroid substrates are similar (1 to 2 μM) and that of its isomerase activity, determined with 5-pregnen 3, 20-dione as a substrate, is 10 μM. The k_cat value determined for its isomerase activity (18.2 min⁻¹) is also higher than that for its dehydrogenase activity (1.4–2.4 min⁻¹). A survey of more than 30 isoflavones and structurally related compounds revealed that daidzein, genistein, biochanin A and formononetin inhibit both the dehydrogenase and isomerase activity of this enzyme. Inhibition is potent and concentration dependent. IC₅₀ values determined for these compounds range from 0.4 to 11 μM, within the plasma and urine concentration ranges of daidzein and genistein of individuals on vegetarian diet or semi-vegetarian diet. These results suggest that dietary isoflavones may exert their biological effects by inhibiting the action of 3β-HSD, a key enzyme of neurosteroid and/or steroid hormone biosynthesis.     

Progestin regulation of 11beta-hydroxysteroid dehydrogenase expression in T-47D human breast cancer cells

This study examined the enzymatic characteristics and steroid regulation of the glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) in the human breast cancer cell line T-47D. In cell homogenates, exogenous NAD significantly increased the conversion of corticosterone to 11-dehydrocorticosterone, while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11β-HSD2 in T-47D cells, while 11β-HSD1 mRNA levels were undetectable. In T-47D cells treated for 24 h with medroxyprogesterone acetate (MPA), 11β-HSD catalytic activity was elevated 11-fold, while estrone (E₁), estradiol (E₂) and the synthetic glucocorticoid dexamethasone (DEX) were ineffective. The antiprogestin mifepristone (RU486) acted as a pure antagonist of the progestin-enhanced 11β-HSD activity, but did not exert any agonistic effects of its own. In addition, RT-PCR analysis demonstrated that MPA was a potent inducer of 11β-HSD2 gene expression, increasing the steady-state levels of 11β-HSD2 mRNA. Taken together, these results demonstrate that 11β-HSD2 is the 11β-HSD isoform expressed by T-47D cells under steady-state conditions and suggest the existence of a previously undocumented mechanism of action of progestins in breast cancer cells.     

Crystallization and preliminary crystal structure of the complex of 17beta-hydroxysteroid dehydrogenase with a dual-site inhibitor

Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1) catalyzes the synthesis of 17β-estradiol (E₂) from estrone, in the ovary and peripheral tissues. While the structures of 17β-HSD1 alone and in complex with E₂ have been determined (D. Ghosh, V. Pletnev, D.-W. Zhu, Z. Wawrzak, W.-L. Duax, W. Pangborn, F. Labrie, S.-X. Lin, Structure of human 17β-hydroxysteroid dehydrogenase at 2.20 Å resolution, Structure 3 (1995) 503–513; A. Azzi, P.H. Rhese, D.-W. Zhu, R.L. Campbell, F. Labrie, S.-X. Lin, Crystal structure of human estrogenic 17β-hydroxysteroid dehydrogenase complexed with 17_β-estradiol, Nature Struct. Biol. 3 (1996) 665–668, no structures of inhibitor/enzyme complex, either modeled or from crystallography, have been reported before the submission of the present paper. The best available inhibitors are among the ‘dual-site inhibitors’, blocking estrogenic 17β-HSD and the estrogen receptor. These compounds belong to a family of estradiol analogues having an halogen atom at the 16 position and an extended alkyl-amide chain at the 7 position (C. Labrie, G. Martel, J.M. Dufour, G. Levesque, Y. Merand, F. Labrie, Novel compounds inhibit estrogen formation and action, Cancer Res. 52 (1992) 610–615). We now report the crystallization of this enzyme/inhibitor complex. The complex of the best available dual-site inhibitor, EM-139, with 17β-HSD1 has been crystallized using both cocrystallization and soaking methods. Crystals are isomorphous to the native crystals grown in the presence of 0.06% β-octyl-glucoside and polyethyleneglycol 4000, with a monoclinic space group C2. Data at 1.8 Å have been collected from a synchrotron source. Even though the size of the inhibitor is greater than that of the substrate, our preliminary X-ray-diffraction study shows that EM-139 fits into the active site in a position similar to that of estrogen. The availability of such structural data will help design more potent inhibitors of estrogenic 17β-HSD.     

平衡施肥对荒漠区黄冠梨生长与品质的影响

对荒漠地区丰产期黄冠梨树生长发育特性、果实和叶片矿质元素含量进行分析,探讨平衡施肥对梨果生长和品质的影响,可为梨园确定合理的施肥水平提供理论依据.选取同一园区12年生黄冠梨为试验材料,设置低氮磷高硫(T₁)、中氮磷中硫(T₂)、高氮磷低硫(T₃)3个处理,以常规施肥为对照(CK).结果表明:不同处理对当年生枝和叶片的生长发育影响不大,但在连续施肥第2年后,T₁促进了当年生枝条的生长,枝条长度和粗度比对照分别提高了16.2%和11.4%.连续施肥2年后可以不同程度提高叶片中矿质元素的含量,其中T₁处理叶片Cu、Fe和Zn含量最高,T₂处理Mg和B含量最高,T₃处理P和Mn含量最高.与对照相比,不同施肥处理对果实产量影响均不明显,但对果实品质有较大影响,处理2年后T₂的可溶性糖和维生素C(Vc)含量均显著增加,分别比对照高出4.2%和7.1%,T₃处理有机酸含量最高,而T₁处理的可溶性固形物、可溶性糖和Vc含量均低于对照.果实中Fe含量与可溶性糖含量、果形指数呈显著正相关,分别与单果质量、有机酸和Vc含量呈显著负相关;P含量与果形指数、果实硬度呈显著正相关,与可溶性糖、有机酸和Vc含量呈显著负相关.综上,T₂处理在保证稳产、丰产的前提下,又起到了改善果实品质的作用,可作为荒漠区黄冠梨生产中适宜的施肥措施.     

Enantiorecognition of triiodothyronine and thyroxine enantiomers using different chiral selectors by HPLC and micro-HPLC

This paper deals with the chiral separation of triiodothyronine (T₃) and thyroxine (T₄) by HPLC and micro-HPLC. The separation of T₃ and T₄ is of great pharmaceutical and clinical interest, since the enantiomers exhibit different pharmacological activities. The HPLC measurements were performed on a chiral stationary ligand-exchange phase using l-4-hydroxyproline bonded via 3-glycidoxypropyltrimethoxysilane to silica gel as a selector. Also a chiral teicoplanin (Chirobiotic ™®) phase was used.

In micro-HPLC the chiral separation behaviour of l-4-hydroxyproline, and of the macrocyclic antibiotics teicoplanin and teicoplanin aglycone was investigated for the enantioseparation of T₃ and T₄. l-4-Hydroxyproline was bonded to 3 μm and the glycopeptide antibiotics were bonded to 3.5 μm silica gel and separations were accomplished by microbore HPLC columns (10 cm × 1 mm I.D.). With both techniques and all chiral selectors investigated T₃ and T₄ were baseline resolved. micro-HPLC was found to be superior to analytical HPLC with respect to low consumption of packing material, mobile phase and analyte.     

Dithioerythritol (DTE) prevents inhibitory effects of triphenyltin (TPT) on the key enzymes of the human sex steroid hormone metabolism

Organotins are known to induce imposex (pseudohermaphroditism) in marine neogastropods and are suggested to act as specific endocrine disruptors, inhibiting the enzyme-mediated conversion of steroid hormones. Therefore, we investigated the in vitro effects of triphenyltin (TPT) on human 5-reductase type 2 (5-Re 2), cytochrome P450 aromatase (P450arom), 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD 3), 3β-HSD type 2 and 17β-HSD type 1 activity. First, the present study demonstrates that significant amounts of TPT occurred in the blood of eight human volunteers (0.17–0.67 μg organotin cation/l, i.e. 0.49–1.92 nmol cation/l). Second, TPT showed variable inhibitory effects on all the enzymes investigated. The mean IC₅₀ values were 0.95 μM for 5-Re 2 (mean of n=4 experiments), 1.5 μM for P450arom (n=5), 4.0 μM for 3β-HSD 2 (n=1), 4.2 μM for 17β-HSD 3 (n=3) and 10.5 μM for 17β-HSD 1 (n=3). To exclude the possibility that the impacts of TPT are mediated by oxidizing essential thiol residues of the enzymes, the putative compensatory effects of the reducing agent dithioerythritol (DTE) were investigated. Co-incubation with DTE (n=3) resulted in dose-response prevention of the inhibitory effects of 100 μM deleterious TPT concentrations on 17β-HSD 3 (EC₅₀ value of 12.9 mM; mean of n=3 experiments), 3β-HSD 2 (0.90 mM; n=3), P450arom (0.91 mM; n=3) and 17β-HSD 1 (0.21 mM; n=3) activity. With these enzymes, the use of 10 mM DTE resulted in an at least 80% antagonistic effect, whereas, the effect of TPT on 5-Re 2 was not compensated. In conclusion, the present study shows that TPT acts as an unspecific, but significant inhibitor of human sex steroid hormone metabolism and suggests that the inhibitory effects are mediated by the interaction of TPT with critical cysteine residues of the enzymes.     

Polyphosphates strongly inhibit the tRNA dependent synthesis of poly(A) catalyzed by poly(A) polymerase from Saccharomyces cerevisiae

Polyphosphates of different chain lengths (P₃, P₄, P₁₅, P₃₅), (1 μM) inhibited 10, 60, 90 and 100%, respectively, the primer (tRNA) dependent synthesis of poly(A) catalyzed poly(A) polymerase from Saccharomyces cerevisiae. The relative inhibition evoked by p₄A and P₄ (1 μM) was 40 and 60%, respectively, whereas 1 μM Ap₄A was not inhibitory. P₄ and P₁₅ were assayed as inhibitors of the enzyme in the presence of (a) saturating tRNA and variable concentrations of ATP and (b) saturating ATP and variable concentrations of tRNA. In (a), P₄ and P₁₅ behaved as competitive inhibitors, with K_i values of 0.5 μM and 0.2 μM, respectively. In addition, P₄ (at 1 μM) and P₁₅ (at 0.3 μM) changed the Hill coefficient (n_H) from 1 (control) to about 1.3 and 1.6, respectively. In (b), the inhibition by P₄ and P₁₅ decreased V and modified only slightly the K_m values of the enzyme towards tRNA.     

Regulation of expression of the 3β-hydroxysteroid dehydrogenases of human placenta and fetal adrenal

The appropriate expression of 3β-hydroxysteroid dehydrogenase/Δ^5→4-isomerase (3β-HSD) is vital for mammalian reproduction, fetal growth and life maintenance. Several isoforms of 3β-HSD, the products of separate genes, have been identified in various species including man. Current investigations are targeted toward defining the processes that regulate the levels of specific isoforms in various steroidogenic tissues of man. High levels of expression of 3β-HSD were observed in placental tissues. It has been generally considered that the multinucleated syncytiotrophoblastic cells are the principal sites of 3β-HSD expression and, moreover, that 3β-HSD expression is intimately associated with cyclic AMP-promoted formation of syncytia. Herein we report the presence of 3β-HSD immunoreactive and mRNA species in uninucleate cytotrophoblasts in the chorion laeve, similar to that in syncytia but not cytotrophoblast placenta. In vitro, 3β-HSD levels in chorion laeve cytotrophoblasts were not increased with time nor after treatment with adenylate cyclase activators, whereas villous cytotrophoblasts spontaneously demonstrated progressive, increased 3β-HSD expression. Moreover, 3β-HSD synthesis appeared to precede morphologic syncytial formation. Thus high steroidogenic enzyme expression in placenta is not necessarily closely linked to formation of syncytia. Both Western immunoblot and enzymic activity analyses also indicated that the 3β-HSD expressed in these cytotrophoblastic populations was the 3β-HSD type I gene product (M_r, 45K) and not 3β-HSD type II (M_r, 44K) expressed in fetal testis. In cultures of fetal zone and definitive zone cell of human fetal adrenal, 3β-HSD expression was not detected until ACTH was added. ACTH, likely acting in a cyclic AMP-dependent process, induced 3β-HSD type II activity and mRNA expression. The higher level of 3β-HSD mRNA in definitive zone compared with fetal zone cells was associated with parallel increases in cortisol secretion relative to dehydroepiandrosterone sulfate formation.     

Immunochemical distribution and immunohistochemical localization of 20β-hydroxysteroid dehydrogenase in neonatal pig tissues

Immunochemical distribution of 20β-hydroxysteroid dehydrogenase (HSD) in neonatal pig tissues was investigated by Western blot analysis of the proteins reacting with anti-20β-HSD antibody. 20β-HSD was present in all organs investigated: brain, lung, thymus, submandibular gland, heart, liver, kidney, spleen, adrenal gland, testis, epididymis, prostate, vas deferens and seminal vesicle. In particular, high concentrations of 20β-HSD were detected in the testis, followed by the kidney and liver, by the [¹²⁵I]-protein A binding method. Immunohistochemical localization of the enzyme was achieved in paraffin sections of the testis, kidney, liver, epididymis, and vas deferens by the streptoavidin-biotin complex method. In the testis, very strong immunostaining was found only in interstitial Leydig cells, whereas the cells in seminiferous tubules, such as Sertoli cells and spermatogenic cells, were entirely negative. In the kidney, strong immunostaining was detected in epithelial cells of Henle's loop. The immunoreactive proteins were also localized in the hepatic lobules of the liver, tall columnar cells of the ductus epididymidis of the epididymis, and mucosal epithelium cells and muscularis of the vas deferens. These observations indicate that tissue distribution of 20β-HSD is similar to that of carbonyl reductase in the human and rat. However, the specific and abundant expression of 20β-HSD in testicular Leydig cells of the neonatal pig, which are concerned with the synthesis of androgens, suggests that 20β-HSD has a very important physiological role in testicular function during the neonatal stage.     

Organization of 3β-hydroxysteroid dehydrogenase/isomerase and cytochrome P450scc into a catalytically active molecular complex in bovine adrenocortical mitochondria

We have previously reported the co-localization [Cherradi et al., Endocrinology 134 (1994) 1358–1364] of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) and cytochrome P450_scc (cyt. P450_scc) in the inner membrane and in the intermembrane contact sites of adrenocortical mitochondria. This observation raises the question of a possible functional association between the two proteins. Isolated bovine adrenocortical mitochondria are able to convert cholesterol to progesterone without the need of exogenous cofactors. An association of 3β-HSD and cyt. P450_scc is observed during the purification of 3β-HSD from mitochondria. The behaviour of 3β-HSD on a column of Heparin-Sepharose is modified by the presence of cyt. P450_scc. Immunoprecipitations from mitochondria with either anti-cyt. P450_scc or anti 3β-HSD antibodies result in a co-precipitation of the two proteins. Both proteins engaged in these immunocomplexes are catalytically active. The interaction was further demonstrated by the surface plasmon resonance method using purified components. An affinity constant of 0.12 μM between 3β-HSD and P450_scc was obtained. These observations suggest that P450_scc and 3β-HSD may associate into a molecular complex in the mitochondrial compartment and may constitute a functional steroidogenic unit, thus opening new possibilities in the regulation of the production of progesterone and its flow in the adrenocortical cell.     

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.     

Dual subcellular localization of the 3β-hydroxysteroid dehydrogenase isomerase: Characterization of the mitochondrial enzyme in the bovine adrenal cortex

The enzyme 3β-hydroxysteroid dehydrogenase isomerase (3β-HSD/I) in an essential step in the biosynthesis of steroid such as progesterone, mineralo- and gluco-corticoids, estrogens and androgens in steroidogenic tissues. It is considered to be mainly localized in microsomes; however, 3β-HSD/I activity has also been described to be associated with mitochondrial preparations. In this study, we examined the subcellular distribution of 3β-HSD/I in bovine adrenocortical tissue and we characterized the catalytic properties of the enzyme present in the various cell compartments. About 30% of the total 3β-HSD/I activity was found to remain tightly associated with the purified mitochondrial pellet. The 3β-HSD/I and 3-ketoreductase activities were found in microsomes as well as in mitochondria. The 3β-HSD/I associated with the mitochondrial fraction did not required addition of exogenous NAD⁺. When the pyridine nucleotide was reduced ollowing addition of substrate of the tricarboxyllic acids cycle, the mitochondrial 3β-HSD/I activity decreased, suggesting that the enzyme utilizes NAD⁺ available from the matrix space. By contrast, the microsomal enzyme was inactive in the absence of exogenous NAD⁺. Submitochondrial fraction disclosed that 3β-HSD/I was associated (i) with the inner membrane and (ii) with a particulate fraction sedimenting in a density gradient between inner and outer membranes. This fraction was characterized as contact sites between the two membranes. 3β-HSD/I specific activity was much higher in this fraction than in the inner mitochondrial membrane. Altogether, these observations suggest that these mitochondrial intermembrane contact sites may represent a spacial organization of functional significance, facilitating both the access of cholesterol to the inner membrane where cytochrome P-450_scc is located and the rapid transformation of its product, pregnenolone, to progesterone, through 3β-HSD/I activity.     

Cytoprotective effects of the antioxidant phytochemical indicaxanthin in beta-thalassemia red blood cells

Antioxidant phytochemicals are investigated as novel treatments for supportive therapy in β-thalassemia. The dietary indicaxanthin was assessed for its protective effects on human β-thalassemic RBCs submitted in vitro to oxidative haemolysis by cumene hydroperoxide. Indicaxanthin at 1.0-10 μM enhanced the resistance to haemolysis dose-dependently. In addition, it prevented lipid and haemoglobin (Hb) oxidation, and retarded vitamin E and GSH depletion. After ex vivo spiking of blood from thalassemia patients with indicaxanthin, the phytochemical was recovered in the soluble cell compartment of the RBCs. A spectrophotometric study showed that indicaxanthin can reduce perferryl-Hb generated in solution from met-Hb and hydrogen peroxide (H₂O₂), more effectively than either Trolox or vitamin C.

Collectively our results demonstrate that indicaxanthin can be incorporated into the redox machinery of β-thalassemic RBC and defend the cell from oxidation, possibly interfering with perferryl-Hb, a reactive intermediate in the hydroperoxide-dependent Hb degradation. Opportunities of therapeutic interest for β-thalassemia may be considered.