PubMed Health⌕ Search

Biomedical subjects

P Sourdaine

Publications and source records attributed to P Sourdaine.

16 recordsLinked to original sources

Aromatase activity modulation by lindane and bisphenol-A in human placental JEG-3 and transfected kidney E293 cells.

Aromatase is the cytochrome P-450 involved in converting androgens to estrogens. The cytochrome P-450 family plays a central role in the oxidative metabolism of compounds including environmental pollutants. Since lindane and bisphenol-A (BPA) are two well-characterized endocrine disruptors that have been detected in animals and humans, it was important to learn whether they could affect aromatase activity and consequently estrogen biosynthesis. The present study investigates the effects of BPA and lindane on cytotoxicity, aromatase activity and mRNA levels in human placental JEG-3 cells and transfected human embryonal kidney 293 cells. Both cell lines were exposed to increasing concentrations of lindane (25, 50 and 75 microM) and bisphenol-A (25, 50 and 100 microM) over different time periods (10 min-18 h). As a result, none of these concentrations showed cytotoxicity. After short pre-incubation times (10 min-6 h), aromatase activity was enhanced by both compounds. Longer time incubation (18 h), however, produced dose-related inhibition. Lindane and BPA had no significant effects on CYP19 mRNA levels. Therefore, lindane and BPA modulate aromatase activity suggesting an interaction with the cytochrome P-450 aromatase. This study highlights the endocrine-modulating properties of lindane and bisphenol-A.

Adult↗

Study of substrate specificity of human aromatase by site directed mutagenesis.

Human aromatase is responsible for estrogen biosynthesis and is implicated, in particular, in reproduction and estrogen-dependent tumor proliferation. The molecular structure model is largely derived from the X-ray structure of bacterial cytochromes sharing only 15-20% identities with hP-450arom. In the present study, site directed mutagenesis experiments were performed to examine the role of K119, C124, I125, K130, E302, F320, D309, H475, D476, S470, I471 and I474 of aromatase in catalysis and for substrate binding. The catalytic properties of mutants, transfected in 293 cells, were evaluated using androstenedione, testosterone or nor-testosterone as substrates. In addition, inhibition profiles for these mutants with indane or indolizinone derivatives were obtained. Our results, together with computer modeling, show that catalytic properties of mutants vary in accordance with the substrate used, suggesting possible differences in substrates positioning within the active site. In this respect, importance of residues H475, D476 and K130 was discussed. These results allow us to hypothesize that E302 could be involved in the aromatization mechanism with nor-androgens, whereas D309 remains involved in androgen aromatization. This study highlights the flexibility of the substrate-enzyme complex conformation, and thus sheds new light on residues that may be responsible for substrate specificity between species or aromatase isoforms.

Amino Acid Sequence↗

Aromatase and breast cancer: W39R, an inactive protein.

BACKGROUND: Aromatase (CYP19) catalyzes the conversion of androgens into estrogens. It is in particular involved in development, reproduction and breast cancer. One of its polymorphisms, W39R localized in the N-terminal region of CYP19, significantly decreases breast cancer risk among Japanese women and was chosen for this study. In this work, we studied the structure-function relationships between W39R polymorphism and CYP19 enzyme activity. OBJECTIVE: To examine the kinetic properties of the mutant W39R recombinant protein in transfected human cells devoid of steroidogenic activity. METHODS: Expression vectors for the wild-type or the mutated R39 aromatase were transiently transfected into E293 human embryonal kidney cells. The conversions of androstenedione to estrone and of testosterone and nortestosterone to 17beta-estradiol were assayed by RIA. Expression of recombinant cDNAs was analyzed by semi-quantitative RT-PCR and immunoblotting. RESULTS: W39R recombinant protein was devoid of aromatase activity whatever the substrate used. This absence of activity was not due to the lack of expression of the recombinant enzyme since the mRNA and protein were detected. CONCLUSION: Our present in vitro study shows that the R39 mutant is unable to synthesize estrogens. This work provides a novel observation, being consistent with the fact that Japanese women with the variant allele (arg) have significantly lower risk of developing a breast tumor.

Alleles↗

Aromatase gene expression in the stallion.

Adult stallion secretes very high estrogen levels in its testicular vein and semen, and the responsible enzyme cytochrome P450 aromatase (P450 arom) is known to be present mainly in Leydig cells. We studied in further details the distribution of equine aromatase in various adult tissues including the brain (hypothalamic area), liver, kidney, small intestine, muscle, bulbourethral gland and testes. The aromatase mRNA was essentially detected by RT-PCR in testis (169+/-14 amol of aromatase mRNA per microg of total RNA) and was barely detectable in brain, or below 0.1 amol/microg RNA in other tissues. This range of expression was confirmed by ELISA (50+/-7 pg/microg total protein) in the testis, and by immunoblot, evidencing a 53 kDA specific protein band in testis and brain only. The corresponding aromatase activity was well detected, by 3H(2)O release from 1beta, 2beta(3)H-androstenedione, in testis and brain (200+/-23 and 25+/-6 pmol/min per mg, respectively) and below 3 pmol product formed/min per mg in other tissues. This study indicates that the testis, among the tissues analyzed, is the major source of aromatase in the adult stallion, and that the aromatase gene expression is specifically enhanced at this level, and is responsible for the high estrogen synthesis observed. Moreover, the study of aromatase in one colt testis has shown lower levels of transcripts, protein and enzyme activity, evidencing that aromatase is regulated during the development and may serve as a useful marker of testicular function. As the second organ where aromatase mRNA and activity are both well detected is brain, this study also underlines the possible role of neurosteroids in stallion on behaviour, brain function or central endocrine control.

Animals↗

New aromatase inhibitors. Synthesis and biological activity of aryl-substituted pyrrolizine and indolizine derivatives.

We report herein the design and the synthesis of some aryl-substituted pyrrolizine and indolizine derivatives, on the basis of a hypothetical pharmacophore structure designed to fit the catalytic site of the human cytochrome P450 aromatase. The in vitro biological evaluation of these compounds allowed us to point out two new potent non-steroidal aromatase inhibitors, MR 20494 and MR 20492, with IC50 values in the range of 0.1 microM.

Aromatase Inhibitors↗

Seasonal variations in sex steroids and male sexual characteristics in Scyliorhinus canicula.

Concentrations of testosterone, progesterone, Delta4-androstenedione, dihydrotestosterone, 11-ketotestosterone, estrone, estradiol-17beta, 5alpha-androstane, 3alpha, 17beta-diol, and 17alpha-hydroxy, 20beta-dihydroprogesterone were determined by radioimmunoassays in the blood plasma and testicular homogenates of Scyliorhinus canicula. Samples were collected almost every month for 27 months. The weights and sizes of reproductive organs and sperm reserves were also measured over the same period. Quantitatively, testosterone was the principal steroid present. Testicular and epididymal weights, sperm reserves, and clasper size varied throughout the year, but not always in a synchronous fashion. Most of the testicular steroids had an annual peak, generally in February, except for progesterone. The plasma concentrations of progesterone, Delta4-androstenedione, and androgens presented various degrees of fluctuation over the year, but were not synchronized: maxima occurred during autumn-winter for androgens and progesterone, during spring for Delta4-androstenedione. Thus, various aspects of S. canicula reproductive function appear to be influenced by season, the sea temperature being, most probably, a major determinant in this respect.

Androstenedione↗

PAAn-1b and PAAn-E: two phosphorothioate antisense oligodeoxynucleotides inhibit human aromatase gene expression.

Estrogen-dependent diseases, especially breast cancers, are frequently treated with aromatase inhibitors. Another more recent strategy is the antisense technology. In this study, after predicting aromatase mRNA secondary structure, we describe the design, the efficiency, and the toxicity of two antisense phosphorothioate oligodeoxynucleotides (PAAn-1b and PAAn-E) directed toward aromatase mRNA. Indeed, 2 microM PAAn-1b and PAAn-E encapsulated with 54 microM polyethylenimine inhibit aromatase activity by 71 and 79%, respectively, in transfected 293 cells, with IC50 values of 0.2 and 0.6 microM. The mechanism of inhibition appears to be specific after using sense and scramble oligodeoxynucleotides as controls and largely decreases aromatase mRNA and protein amounts. Moreover, PAAn-1b and PAAn-E are not cytotoxic for 293 cells. This study finally provides a new strategy for aromatase inhibition. It offers new tools for studying aromatase gene expression and its role in cancer for instance, and this could be of help for the therapy of estrogen-dependent diseases.

Actins↗

Regulation of aromatase activity within the breast.

Local oestrogen biosynthesis within the breast can be highly variable, in vitro aromatase activity both in breast cancers and mammary adipose tissue displaying over a 40-fold range between the highest and lowest levels. Evidence is presented to show that: (i) transcriptional activity may influence oestrogen biosynthesis within breast cancers in that both aromatase mRNA and STAT nuclear binding are correlated positively to in vitro aromatase activity; (ii) the local presence of cancer may enhance aromatase activity in particulate fractions and primary fibroblast cultures from mammary adipose tissue; (iii) tumour extracts and breast cyst fluids may induce aromatase in cultured fibroblasts, the active principles responsible for these effects being incompletely defined (although the combination of interleukin (IL)-6 and its soluble receptor dramatically enhances aromatase activity, it is unclear whether this particular cytokine system can account for the stimulatory effects of breast extracts and fluids); (iv) the aromatase activities in both breast cancer and adipose tissues are susceptible to classical aromatase inhibitors such as aminoglutethimide and 4-hydroxyandrostenedione (and to newer inhibitors such as CGS16949 and CGS20267 at low nanomolar concentrations) but reduced sensitivity to 4-hydroxyandrostenedione may be observed in certain breast cancers. These findings may have important implications for the development and progression of hormone-dependent cancers within the breast.

Adipose Tissue↗

Aromatase activity and CYP19 gene expression in breast cancers.

The aromatase enzyme complex is responsible for the conversion of C19 androgens to oestrogens. Aromatase expression in oestrogen-responsive breast cancers may be an important mechanism of autocrine regulation in tumour growth. To evaluate whether aromatase cytochrome P450 (P450arom) transcript levels within breast tumours were correlated to the enzyme activity, a specific competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed. In this reaction, a 32 base-deleted complementary RNA was used as internal standard. In vitro aromatase activity was measured by either the tritium release assay or characterization of oestrogen fractions. Results indicate that there is a positive correlation between P450arom transcript levels and enzyme activity, but the relationship does not reach statistical significance. Therefore, whereas aromatase mRNA quantification may be an option by which to monitor the potential of tumour to synthesize oestrogens, it will not accurately reflect enzyme activity in a minority of tumours. Preliminary evidence was obtained in a tumour with low enzyme activity and a high P450arom transcript level for the presence of an endogenous aromatase inhibitor. This study highlights the necessity to characterize factors involved in the regulation of aromatase activity in such tumours.

Aromatase↗

Cell-cell interactions in the testis of teleosts and elasmobranchs.

In this paper we present the state of knowledge on cell-cell interactions in the testis of two groups of anamniote vertebrates--teleosts and elasmobranchs--which include most fish. In these fish, the structural organization of the testis differs fundamentally from that which characterizes amniotes in which the germinal tissue is located in tubules open at both ends and consists of a permanent population of Sertoli cells associated with successive stages of germ cell development. In fish, the spermatogenic unit of testis is the spermatocyst, which corresponds to one germ cell or to a clone of isogenetic germ cells, enclosed by one or several Sertoli cells, which form the wall of the cyst. In fish testis, the Sertoli cells do not represent a permanent population of cells. Although both are of the cystic type, the teleost and elasmobranch testes are differently organized. In elasmobranchs, primary spermatogonia and Sertoli cells lie initially free within the interstitial tissue, before becoming sequestered by a basement membrane; the testis is then composed of a mass of spermatocysts which contain many Sertoli cells, each being associated with a clone of germ cells. In contrast, in teleosts, the cysts are confined to large elongated structures limited by a basement membrane. These structures are either lobules originating under the albuginea or tubules which, in contrast to those of mammals, are anastomosed. In the lobules, the spermatocysts start to develop at the blind end of the lobules and migrate towards the efferent system, whereas in the tubules, the spermatocysts are located against the basement membrane, all along the tubules and do not migrate. In elasmobranchs, unlike teleosts, Leydig cells are either absent from the interstitial tissue or rare and undifferentiated and their role in steroid production is at best marginal. While many studies have focused on topographical and functional interactions between the diverse cell types present in mammalian testis, only a few studies have brought particular attention to these aspects in fish. In fish, like in mammals, testicular cell-cell interactions are based on structural elements and chemical factors. Occasionally, various adhering junctions have been observed, essentially in teleosts, between Sertoli cells, between Sertoli cells and germ cells, between germ cells themselves, and interstitial cells. Furthermore, in some teleost species, using horseradish peroxidase or lanthanum salts, the presence of tight junctions between Sertoli cells has been correlated to the occurrence of a Sertoli barrier. In these species, the barrier develops after meiosis so that only haploid germ cells are shielded from the vascular system. In fish, recent development of techniques which enable the preparation and in vitro culture of enriched populations of testicular cells and of spermatocysts, has allowed investigations on functional aspects of cell-cell interactions. In particular, data have been obtained, in the trout, on the control of spermatogonia proliferation by Sertoli cell-conditioned media and, in the dogfish, on the steroidogenic activity of Sertoli cells, in relation to the differentiation stage of the associated germ cells. Furthermore information exists, in the trout, showing that intratubular macrophages may participate in the re-initiation of spermatogonial proliferation. In conclusion, the cytoarchitecture of fish testis, as compared to that of mammals, presents original features which provide unique opportunities to develop fruitful studies for a better understanding of the complex control mechanisms underlying testicular function in vertebrates.

Animals↗

Analysis of the aromatase cytochrome P450 gene in human breast cancers.

Aromatase activity may be detected in most, but not all, breast cancers, and in certain tumours there appears to be decreased sensitivity to the aromatase inhibitor 4-hydroxyandrostenedione (4-OHA). The aims of the present study were to measure aromatase activity, and its sensitivity to 4-OHA, in breast tumours, and to examine the CYP19 gene encoding the aromatase cytochrome P450 (P450arom) for the presence of mutations. In vitro aromatase activity and sensitivity to 4-OHA were measured by determining the conversion of tritiated testosterone to tritiated oestradiol in breast tumour tissue in the absence and presence of 4-OHA (10 nM). Genomic DNA was extracted from five tumours: one showing no detectable aromatase activity and four displaying evidence of aromatase activity (two sensitive and two insensitive to 4-OHA). Subsequent PCR-single-strand conformation polymorphism analysis revealed a variation in the mobility of single-stranded DNA for exons III, VII and X, corresponding, as shown by direct sequencing of PCR products, to common polymorphism of the aromatase gene. This study does not provide evidence for mutation in the coding exons of the P450arom gene which would account for either the absence of aromatase activity or its changed sensitivity to 4-OHA in breast cancers.

Androstenedione↗

Stage-dependent modulation of Sertoli cell steroid production in dogfish (Scyliorhinus canicula).

Seminiferous lobules of dogfish (Scyliorhinus canicula) testis comprise cysts formed by steroid-producing Sertoli cells associated with germ cells at an identical stage of spermatogenesis. These lobules were isolated in four populations corresponding to lobules with spermatogonia (A), spermatocytes (B), early spermatids (C) and late spermatids (D). They were used for steroid radioimmunoassay or incubated with 22 alpha-hydroxycholesterol or with dibutyryl cyclic AMP (dibutyryl cAMP) to measure steroid production. Our results indicate that progesterone was the major steroid in seminiferous lobules at all stages of spermatogenesis except in lobules A. Furthermore, marked changes in the distribution of steroids were observed according to the stage of spermatogenesis; progesterone, 4-androstenedione, testosterone and 17 alpha-hydroxy,20 beta-dihydroprogesterone concentrations were highest in lobules D, whereas dihydrotestosterone concentrations decreased during spermatogenesis. No significant stage-related change was observed for 3 alpha-diol and 3 beta-diol. Incubation experiments revealed that the isolated seminiferous lobules at all stages can synthesize steroids from hydroxycholesterol and that lobules D have the highest basal contents of androstenedione and testosterone. Furthermore, when dibutyryl cAMP and 10 mumol hydroxycholesterol l-1 were added together to the cultures, an enhancement of the steroid secretion was observed rather than a change in synthesis. Our results also indicated that the responsiveness of the lobules to dibutyryl cAMP varies according to the stage of spermatogenesis and to the steroid assayed. Overall, this study indicated that germ cells probably markedly influence Sertoli cell steroidogenesis in the adult dogfish testis.

Androgens↗

The adult dogfish (Scyliorhinus canicula L.) testis: a model to study stage-dependent changes in steroid levels during spermatogenesis.

The unit structure of the testis of Scyliorhinus canicula L. is the seminiferous lobule arranged in topographically distinct zones and comprised of cysts formed by steroid-producing Sertoli cells associated with germ cells at an identical stage of spermatogenesis. In this experiment, testicular tissues from different dogfish corresponding to the zones of lobules with spermatogonia (A), spermatocytes (B), early spermatids (C) and late spermatids (D) were dissected out and pooled. They were either used for steroid radioimmunoassay or incubated to measure steroid production in response to dogfish pituitary extract or to dibutyryl cyclic AMP (dbcAMP). Our results indicate that testosterone is the main testicular androgen in this species. Furthermore, marked changes in the distribution of steroids were observed according to the zone of spermatogenesis, testosterone concentrations being highest in zones A and D. The relative contents of this hormone (steroid levels normalized to an equivalent number of Sertoli cells) as well as of delta 4-androstenedione and of progesterone increased from A to D. The concentrations of delta 4-androstenedione and progesterone were highest in zone D. Relative contents of 5 alpha-androstane-3 alpha,17 beta-diol, dihydrotestosterone and 11-ketotestosterone were very low in A when compared with the other zones. Finally, whereas no significant stage-related variation in the concentration of oestradiol-17 beta and 17 alpha-hydroxy, 20 beta-dihydroprogesterone was observed, the relative content of these steroids was lowest in zone A. Incubation experiments revealed that dogfish pituitary extract and dbcAMP can stimulate androgen synthesis (about twofold) and that a stage-related change of steroid responsiveness to dbcAMP exists (only zones A and D were responsive). Overall, the results of this study indicate that germ cells most probably influence Sertoli cell steroidogenesis and responsiveness in the adult dog fish testis.

Androgens↗

MR 20492 and MR 20494: two indolizinone derivatives that strongly inhibit human aromatase.

In this study, we describe the synthesis of a new family of indolizinone derivatives designed to fit an extrahydrophobic pocket within the active site of aromatase and to strongly inhibit human aromatase. This could help improve the specificity of the inhibitors. Equine aromatase, very well characterized biochemically, is used as a comparative model. Indeed, in a previous comparison between both human and equine aromatases, we described the importance of the interaction between the inhibitor and this pocket for the indane derivative MR 20814. MR 20492 and MR 20494 are more potent inhibitors of human aromatase (Ki/Km: 1.0+/-0.3 and 0.5+/-0.3, respectively). The Ki/Km for MR 20494 is slightly higher than that obtained for fadrozole (0.1+/-0.0) and Ki/Km for both indolizinone derivatives are lower than those obtained for 4-hydroxyandrostenedione (1.9+/-0.8) and MR 20814 (8.1+/-.7). These new compounds are not enzyme inactivators. Moreover, as indicated by the higher Ki/Km values obtained with equine enzyme (9.0+/-0.6 and 6.1+/-1.6 for MR 20492 and MR 20494, respectively), both human and equine aromatase active sites appear to be structurally different. Difference absorption spectra study (350-500 nm) revealed that MR20492 and MR20494 were characterized by a combination of type-I and -II spectra with both enzymes. This result could be due to the isomerization of the molecule in polar solvent (Z and E forms). The evaluation of these new molecules, as well as 4-hydroxyandrostenedione and fadrozole, on aromatase activity in transfected 293 cell cultures evidenced a strong inhibition (IC50: 0.20+/-0.03 microM, 0.20+/-0.02 microM and 0.50+/-0.40 microM for MR 20494, fadrozole and 4-OHA, respectively) except for MR 20492 (3.9+/-0.9 microM) and MR 20814 (10.5+/-0.6 microM). These results proved that these molecules formed part of a promising family of potent inhibitors and that they penetrate 293 cells, without evidencing any cytotoxicity in Hela cells with MTT assay. This is thus encouraging for the development of new drugs for the treatment of estrogen-dependent cancers, these molecules also constitute new tools for understanding the aromatase active site.

Animals↗