Exposure to individual polycyclic aromatic compounds impairs the cardiac performance of American lobster (Homarus americanus) larvae.

The potential for oil spills poses a threat to marine organisms, the toxicity of which has been attributed primarily to polycyclic aromatic compounds (PACs). Predictive tools such as the target lipid model (TLM) have been developed to forecast and assess these risks. The aim of the present study was to characterize the cardiotoxicity of 10 structurally diverse PACs in American lobster (Homarus americanus) larvae by assessing heart rate following a 48 h exposure in a passive dosing system, and subsequently use the TLM framework to calculate a critical target lipid body burden (CTLBB) for bradycardia. Exposure to 8 of the 10 PACs resulted in concentration-dependent bradycardia, with phenanthrene causing the greatest effect. The TLM was able to effectively characterize bradycardia in American lobsters, and the cardiotoxic CTLBB value determined in this study is among the most sensitive endpoints included in the CTLBB database. This study is one of the first to apply the TLM to a cardiac endpoint and will improve predictive models for assessing sublethal impacts of oil spills on American lobster populations.

Switching agonistic, antagonistic, and mixed transcriptional responses to 11 beta-substituted progestins by mutation of the progesterone receptor.

The study of transcription activation by a series of RU486-related 11 beta-substituted progestins revealed three types of ligands: agonists, antagonists, and a novel type of compounds that exerted a mixed activity. These ligands conferred to the human progesterone receptor (hPR) only weak activation properties despite high affinity binding and, hence, acted as agonists and, at the same time, as partial antagonists of pure agonists. When the same series of ligands was tested with mutant PRs, transcriptional activation/inactivation profiles were different from those seen with the wild-type PR, since several steroids initially classified as antagonists switched to mixed responses. One compound that acted as an antagonist for the hPR was an agonist for a mutated PR in which 15 amino acids of the hormone-binding domain were replaced by the corresponding divergent residues of the chicken homolog. In analyzing a series of steroids with wild-type and mutant PRs, we observed that a phenyl group (or a phenyl derivative) in the 11 beta position of RU486-related steroids generates antagonism with hPR, but has to be bound in a critical position in the hormone-binding domain to exert its antagonistic activity. Apparently, a deviation from this positioning by mutations in the hormone-binding domain can generate mixed or even agonistic activities.

Residues in the ligand binding domain that confer progestin or glucocorticoid specificity and modulate the receptor transactivation capacity.

To localize regions conferring ligand binding specificity of the human glucocorticoid (hGR) and progesterone (hPR) receptors, we constructed chimeras comprising the DNA-binding domain of the yeast transcription factor GAL4, linked to the ligand binding domain of hGR or hPR. Replacement of a sequence of hGR encompassing helices H6 and H7 with the homologous sequence from hPR creates a chimeric protein GP3, which binds the progestin RU 27987 with high affinity, and results in a concomitant loss of glucocorticoid binding [dexamethasone (DEX), RU 43044]. Moreover, GP3 is not able to mediate RU 27987-induced transactivation. A detailed mutational analysis of this sequence and the study of the recently solved hPR crystal structure revealed five residues that confer progestin responsiveness to GR or modulate ligand binding and transcriptional activation. Notably, the simultaneous presence of residues Ser637 and Phe639 on GP3, lining the ligand binding pocket, is specifically involved in RU 27987 binding. The absence of residues Asp641, Gln642, and Leu647 on GP3 is accountable for the lack of glucocorticoids binding on GP3. Unlike residue 642, residues 641 and 647 are not in direct contact with the ligand and most likely act through steric-mediated interactions. The presence of Gln642 and Leu647 are determinant for transcriptional activation in response to DEX and RU 27987, respectively. DEX-dependent transactivation is further enhanced by the presence of Leu647.

Properties of the cytoplasmic progestin-binding protein in the rabbit uterus.

An exchange assay for the measurement of total cytoplasmic progestin binding sites has been developed on rabbit uterine cytosol using the highly potent progestin, R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) labelled to a high specific activity. This compound has several advantages over progesterone: it is not bound by plasma corticosteroid binding globulin; it has high affinity for the progestin receptor; it binds virtually as fast as progesterone to the receptor, but the complex formed dissociated 8 times slower; its binding is not displaced by more than 2% by compounds devoid of progestational activity (estrogens, testosterone, dexamethasone, aldosterone). Bound endogenous progesterone was exchanged by tritiated R 5020 in a time compatible with receptor stability. At 0 C, total exchange of filled sites occurred in less than 4 h; at this temperature the R 5020-receptor complex was stable for at least 28 h. The conformation of the R 5020-receptor complex was investigated in sucrose density gradients under various experimental conditions. Unlike progesterone, it was possible to detect a 7S peak in uterine cytosol obtained from rabbits injected with a tracer dose of [3H]R 5020 1 h prior to sacrifice.

Psychosocial aspects of neoplastic disease: II. Affective and cognitive effects of chemotherapy in cancer patients.

A battery of cognitive and affective tests administered to 50 consecutively admitted medical oncology patients revealed cognitive impairment to be a common occurrence in the absence of affective disorders or other psychopathology. Chemotherapy was the major variable associated with cognitive impairment in these patients. These findings suggest that the consultant psychiatrist should be aware of chemotherapy as a possible source of behavioral change and emotional distress in cancer patients.

Self-inhibition of steroid secretion by amphibian adrenocortical cells is not mediated through glucocorticoid receptors.

To investigate a possible direct action of glucocorticoids on adrenal steroidogenesis, the effect of corticosterone on the conversion of pregnenolone into various metabolites by frog adrenal tissue was examined. Frog interrenal slices were incubated with [3H]pregnenolone (1 mCi/ml) and the various labelled metabolites analysed by reverse-phase high-performance liquid chromatography. With the methanol gradient used, five identified steroids were resolved: progesterone, 11-deoxycorticosterone, corticosterone, 18-hydroxycorticosterone and aldosterone. Corticosterone (10 micrograms/ml) induced a 45-80% decrease in all steroids synthesized from [3H]pregnenolone. In contrast, the glucocorticoid agonist dexamethasone did not reduce the rate of conversion of pregnenolone into its metabolites. In addition, the inhibitory effect of corticosterone was not reversed by the specific glucocorticoid antagonist RU 43044. These results show that corticosterone exerts a direct inhibitory effect on adrenal steroid secretion. In addition, our data indicate that the ultra-short regulation induced by corticosterone is not mediated through glucocorticoid receptors.

Sequences in the ligand-binding domains of the human androgen and progesterone receptors which determine their distinct ligand identities.

The natural ligands of the progesterone (PR) and androgen (AR) receptors, progesterone and testosterone, differ only by their 17 beta-substitution. To identify within the AR and PR ligand-binding domains (LBDs) the sequences responsible for the differential recognition of these ligands, chimeric LBDs assembled from five homologous AR/PR 'cassettes' linked to the GAL4-DNA binding domain were constructed, and their ligand binding and transactivation characteristics were determined. Replacing the central cassette 3 of PR by that of AR generated a progesterone- and testosterone-responsive PR LBD with the AR residues 788-RHLS-791 being specifically involved in testosterone recognition, while the introduction of the C-terminal PR cassette 5 into AR conferred progestin responsiveness onto the AR LBD. These results suggest that residues within AR 788-RHLS-791 interact with the testosterone 17 beta-OH, while PR cassette 5 apparently contains the amino acid(s) specifically involved in the recognition of the progesterone 17 beta-acetyl group. However, ligand binding and transactivation by these chimeras were significantly decreased compared with those of the parental LBDs, indicating that residues located outside of these cassettes contribute to the proper positioning of the steroids in the AR and PR ligand-binding pockets (LBPs). Indeed, certain AR/PR chimeras acquired efficient ligand binding, but were unable to transactivate, indicating that the ligand was improperly bound in the chimeric. LBP and could not induce the conformational changes leading to a transcriptionally competent activation function (AF-2) within the LBD. The properties of the various LBD chimeras are discussed in view of the recently solved three-dimensional structures of the retinoid X receptor alpha apo- and retinoic acid receptor gamma holo-LBDs.

Synthesis and evaluation of a new series of mechanism-based aromatase inhibitors.

A series of new 4-(alkylthio)-substituted androstenedione analogues was designed as potential suicide inhibitors of aromatase on the basis of mechanistic considerations on the mode of action of the enzyme. Their synthesis and biological evaluation are described. Among the most interesting are the 4-[(difluoromethyl)thio]-, 4-[(fluoromethyl)thio]-, and 4-[(chloromethyl)thio]androstenediones 12, 13, and 14 with respective IC50's of 2.7, 0.8, and 0.94 microM. Compound 12 was a reversible inhibitor of aromatase while compounds 13 and 14 displayed time-dependent kinetics of inhibition with respective KI's and half-times of inactivation of 30 nM and 3.75 min for 13 and 30 nM and 3 min for 14. The inhibition of aromatase by 14 was NADPH-dependent, and was protected by the presence of substrate (0.5-1 microM), while beta-mercaptoethanol (0.5 mM) failed to protect the enzyme from inactivation. Dialysis failed to reactivate aromatase previously inactivated by 14. The mechanistic implications of these findings are discussed.

Structure-activity relationships of a new family of steroidal aromatase inhibitors. 1. Synthesis and evaluation of a series of analogs related to 19-[(methylthio)methyl]androstenedione (RU54115).

During the course of a study aimed at the search for new potent aromatase inhibitors, several new androstenedione analogs were synthesized and evaluated. This study led to the discovery of 19-[(methylthio)methyl]androsta-4,9(11)-diene-3,17-dione (7; RU54115) already described by our laboratory. The object of the present series of papers is to disclose the result of the structure-activity relationship studies that gave rise to this compound. This first part deals mainly with the substitution in the 19-position of the steroid nucleus. Several parameters were varied, the length of the chain and its rigidity and branching, as well as the nature of the heteroatom itself and its substitution. The interaction of these new compounds with human placental aromatase in competition with the substrate androstenedione was studied by difference visible spectroscopy. The in vivo aromatase-inhibiting activities were evaluated by measuring the estradiol lowering after oral administration of the compounds to PMSG-primed female rats.

RU 3117 a steroidal compound with high affinity for sigma sites in rat testis membranes.

RU 3117 belongs to a new series of steroids which exhibited a high relative binding affinity (RBA) for (+)[3H]PPP sites in rat testis membranes; its RBA was about 40 times higher than that of progesterone. Furthermore, it is devoid of any binding to classical steroid receptors; therefore in order to study its binding parameters on rat testis membranes it was tritiated. [3H]RU 3117 bound at least two distinct sites with Ka values of 0.4 +/- 0.06 x 10(9) M(-1) and 1.3 +/- 0.2 x 10(7) M(-1). Using this marker, competition studies with cold haloperidol showed that a part of this binding was haloperidol-sensitive, whereas another part was haloperidol-resistant. Interestingly, progesterone described as a sigma ligand competes with [3H]RU 3117 binding, with an RBA of 1.6%. When haloperidol was preincubated (250 nM) with rat testis membranes, in order to mask the sigma sites, we observed that DTG (1,3-di-O-tolylguanidine) and haloperidol displayed a very low RBA (< 0.1%) and were not able totally to displace the [3H]RU 3117 binding up to 50 microM. Furthermore, benztropine exhibited a significant RBA of 19% but its displacement curve showed a plateau (500-50,000 nM). These results showed that part of the haloperidol-resistant sites was benztropine sensitive but another part was displaced neither by haloperidol nor by benztropine. The presence of these remaining binding sites was confirmed by preincubating a mixture of haloperidol and benztropine with testis membranes. Under these conditions, [3H]RU 3117 displayed a Ka of 1.0 +/- 0.01 x 10(7) M(-1), and we observed that these sites were recognized, up to now, only by the steroids RU 1968 and RU 54173 which are also devoid of any binding to classical nuclear steroid receptors.

11 beta-Amidoalkoxyphenyl estradiols, a new series of pure antiestrogens.

11 beta-Amidoalkoxyphenyl estradiols, a series of new antiestrogens, have been prepared and compared with tamoxifen (TAM) and 4-hydroxytamoxifen (OH-TAM). In vitro, these compounds were up to 20 times as active as OH-TAM on estradiol (E2)-stimulated MCF-7 cells. Unlike TAM or OH-TAM which were inactive, they displayed potent growth inhibitory effects on MCF-7 cells stimulated by a cocktail of epidermal growth factor and platelet derived growth factor. One of the most active compounds, 5e, was tested in vivo for its antiuterotrophic and antitumoral activities: it proved to be fully antiuterotrophic at 3 mg/kg subcutaneously in mice while being devoid of any uterotrophic activity. It inhibited the E2-induced growth of MCF-7 tumors implanted in nude mice and prevented the partial agonistic activity of TAM on MCF-7 tumor growth in ovariectomized mice. Moreover, on MCF-7 variant tumors, 5e, unlike TAM, did not display any proliferative activity, but inhibited the TAM-induced growth. Overall, these results show that this new series of compounds displays an improved activity profile compared with that of TAM, on tests relevant to human breast cancer treatment.

RU 58841, a new specific topical antiandrogen: a candidate of choice for the treatment of acne, androgenetic alopecia and hirsutism.

A new topically active non-steroidal antiandrogen, RU 58841 has been synthesized. It displays high affinity for the hamster prostate and flank organ (F.O.) androgen receptors. In vivo, when topically applied, it exerts a potent dose-dependent regression of F.O. area at a dose as low as 1 microgram/animal while being devoid of antiandrogenic activity on deep accessory sex organs and of any effect on testosterone level up to 100 micrograms/animal. In the same species, after subcutaneous administration, it induces at the dose of 300 micrograms/animal, a small decrease in F.O. area equivalent to that of 1 microgram applied topically and a weak systemic activity. In intact rats, no effects were observed up to 1 microgram/animal whatever the route of administration. These results suggest that RU 58841 might useful for the topical treatment of androgen-dependent skin disorders such as acne, androgenetic alopecia and hirsutism.

Preliminary pharmacokinetics and metabolism of novel non-steroidal antiandrogens in the rat: relation of their systemic activity to the formation of a common metabolite.

The non-steroidal antiandrogens, RU 58841 and RU 56187 are amongst the most active of a new series of N-substituted aryl hydantoins or thiohydantoins. Their pharmacokinetics and principal metabolic profiles have been evaluated in rat plasma after intravenous administration of a 10 mg/kg dose. Both compounds disappear relatively rapidly from the plasma (elimination half-life of the order of 1 h), but they form a common metabolite, the N-desalkyl derivative, RU 56279, which is eliminated much more slowly. The percentage transformations of each into RU 56279, estimated from the AUCs of the metabolite compared with the AUC obtained after administration of RU 56279 itself, were respectively 1% and 77%. In parallel, their in vivo activity, as well as that of their metabolites, was determined with respect to parameters related to systemic antiandrogenic effects (prostate and seminal vesicle weights). The results showed that: (1) the common metabolite, RU 56279, is clearly antiandrogenic; (2) there appears to be a relationship between the percentage formation of this metabolite and the systemic antiandrogenic activity of the compounds. Thus, the pharmacological profile of RU 58841 which displays a potent local antiandrogenic activity without systemic effects can be related to its very low propensity to form the N-desalkyl metabolite.

11 beta-amidoalkyl estradiols, a new series of pure antiestrogens.

In order to find new antiestrogens, devoid of any agonistic activity, a series of 11 beta-amidoalkyl estradiols were prepared. These compounds have been studied in comparison with tamoxifen (TAM): in vitro, for their relative binding affinities (RBA) for mouse and MCF-7 estrogen receptors (ER) and for their antiproliferative effect on MCF-7 (estradiol or EGF/PDGF stimulated) and Ly2 human breast cancer cell lines; in vivo, for their uterotrophic/antiuterotrophic activities in the mouse and for their antitumoral activities on MCF-7 tumors implanted in nude mice. The most representative compounds are N-methyl-N-isopropyl-(3,17 beta-dihydroxy-estra-1,3,5(10)-trien-11 beta-yl)- undecanamide (RU 51625) and its 17 alpha-ethynyl derivative (RU 53637). They showed good RBAs for ER and a stronger antiproliferative effect than TAM in vitro. Unlike TAM, these compounds inhibited growth factor stimulated MCF-7 proliferation, and the growth of the TAM resistant cell line Ly2. In vivo, they were completely devoid of uterotrophic activity, when given subcutaneously in mice, but exhibited a slight agonistic effect when administered orally. They showed interesting antitumor activities in nude mice by the percutaneous route, but RU 53637 was significantly more potent than RU 51625 when given orally.

RU 58668: further in vitro and in vivo pharmacological data related to its antitumoral activity.

Previous studies with the pure antiestrogen RU 58668 showed that this compound proved to be highly antiproliferative in vitro, and to be the only antiestrogenic compound so far known to induce long-term regression of MCF-7 tumours implanted into nude mice. In order to obtain more insight into the therapeutic potential of this molecule, we performed a new set of experiments in vitro and in vivo in comparison with tamoxifen and/or ICI 182,780. In vitro, 1 nM RU 58668 induced an accumulation of MCF-7 cells in G0/G1 phases of the cell cycle within 48 h and, in contrast to trans-4-hydroxy-tamoxifen, blocked the invasiveness of ras-transfected MCF-7 cells into the chick embryo heart during the three weeks of co-culture. An in vivo dose-effect relationship study showed that RU 58668 induced a regression of MCF-7 tumour with as low a dose as 10 mg/kg/week, and that such an effect can not be obtained either with a sublethal dose of adriamycin or with ICI 182,780, (2-250 mg/kg/week). This reduction in the tumour volumes accords with histological modifications of the tumours, which showed a decrease in the ratio of epithelial cells over the tumoral mass, and with a concomitant decrease in their regrowth potential when reimplanted into naive nude mice. Taken together, these results suggest a promising usefulness for RU 58668 in the treatment of metastatic breast cancer in women.

RU 58,668, a new pure antiestrogen inducing a regression of human mammary carcinoma implanted in nude mice.

RU 58,668, a new steroidal antiestrogen, has been synthesized. Its in vitro and in vivo pharmacological activities have been compared to those of tamoxifen and ICI 182,780. In vitro, it displayed affinities for human and murine estrogen receptors equivalent to those of 4-hydroxy-tamoxifen, along with moderate affinities for progestin and glucocorticoid receptors. RU 58,668 proved to be a potent antiproliferative agent on MCF-7 cells stimulated by estradiol, or by exogenous or endogenous growth factors (IC50, 0.01 nM). It also inhibited the growth of the insulin-stimulated T47D cell line. In vivo, RU 58,668 displayed a total anti-uterotrophic activity in mice or rats without exhibiting any agonistic effect. Moreover, RU 58,668 was the only antiestrogenic compound tested so far to be able to induce a long term regression of human mammary MCF-7 tumors implanted in nude mice, suggesting its potential use for the treatment of advanced breast cancer.

Non-steroidal antiandrogens: synthesis and biological profile of high-affinity ligands for the androgen receptor.

New N-substituted arylthiohydantoin antiandrogens were synthesized. These compounds presented exceptionally high relative binding affinities (RBAs) for the rat androgen receptor (AR): up to 3 times that of testosterone (T) and 100 times the RBAs of non-steroidal antiandrogens such as flutamide, Casodex and Anandron. Furthermore, unlike available markers for AR, they were totally devoid of any binding to the other steroid receptors. RU 59063, the molecule with the highest RBA, was tritiated. When it was compared to [3H]T for the assay of rat, mouse, hamster and human AR, it gave rise to the same number of binding sites but its K alpha (6 x 10(9) M-1) for rat and human AR were, respectively 3 and 8 times higher than that of T. Moreover RU 59063, unlike T, was devoid of any specific binding to human plasma. In vivo, these compounds displayed antiandrogenic activity while being devoid of any agonistic effect. Thus, RU 56187, given orally in castrated male animals, prevented in a dose-dependent manner the effects of 3 mg/kg testosterone propionate (TP) on mouse renal ornithine decarboxylase (acute test) and of 0.5 mg/kg TP on rat prostate weight (chronic test). In these two models, its ED50 was 0.6 and 1 mg/kg, respectively. In the intact rat, when given alone, it inhibited dose-dependently the effect of endogenous androgens on the seminal vesicles (ED50 approximately 1 mg/kg) and prostate (ED50 approximately 3 mg/kg) weights. These results suggest that these new compounds may be useful as specific markers for the androgen receptor as well as for the treatment of androgen-dependent diseases or disorders such as prostate cancer, acne, hirsutism and male pattern baldness.

Pharmacological profile of RU 58642, a potent systemic antiandrogen for the treatment of androgen-dependent disorders.

The pharmacological profile of RU 58642, a new non-steroidal antiandrogen was investigated both in vitro and in vivo. In vitro, the compound displays a strong and specific affinity for androgen receptor. In vivo, its antiandrogenic activity was evaluated in castrated rat supplemented with testosterone propionate and in intact animals on prostate, seminal vesicles weight and serum levels of testosterone by oral and subcutaneous route. In castrated rats RU 58642 induced a significant decrease in prostate weight at a dose as low as 0.3 mg/kg whatever the route of administration. In intact rats its activity was compared to that of other non-steroidal antiandrogens such as flutamide, nilutamide and bicalutamide. RU 58642 proved to be significantly more potent than the reference compounds in reducing prostate weight: 3-30 times orally and 3-100 times subcutaneously, and thus the most potent antiandrogen to date to our knowledge. These results suggest that this compound may be very useful in the treatment of systemic androgen-dependent diseases.

Exploration of the therapeutic potential of the antiestrogen RU 58668 in breast cancer treatment.

The recently described pure antiestrogen RU 58668 displayed potent antiproliferative activities in vitro on several ER+ human mammary cell lines, stimulated either by estradiol or by endogenous or exogenous growth factors. Moreover, when administered to nude mice it proved to be the only antiestrogen to induce regression (at least 10 weeks) of estradiol-stimulated MCF-7 tumors, whereas tamoxifen only stabilized the tumor volume for 4 to 8 weeks. So the first purpose of this work was to study the effect of RU 58668 for 6 months and to evaluate its activity on tumors which escaped from the tamoxifen treatment. On the other hand, we looked for its effect on models more related to frequently described clinical observations, such as the overexpression of an oncogene or the implication of autocrine or paracrine growth factors. Long-term studies of RU 58668 on the estradiol-stimulated MCF-7 model showed that this compound induced a shrinking of tumor volumes for at least 25 weeks (3 to 6 times longer than the stabilization induced by tamoxifen) and was able to reduce the volume of tumors which escaped from, or even were stimulated by, tamoxifen. On models of spontaneously growing tumors, where the overexpression of an oncogene or the production of growth factors was involved, RU 58668 induced the same tumor shrinking that was previously observed on estradiol- or tamoxifen-stimulated models. Finally, when MCF-7 cells were injected in the uteri, a spontaneous tumor take was observed (in about 80-90% of the animals), leading to a more than twofold increase in uterus weight. This growth is largely stimulated by estradiol and tamoxifen. On this model, histological examination showed that only 30% of the animals receiving RU 58668 displayed tumoral microfoci. These studies suggest that RU 58668 may be used for the treatment of ER+ patients which are primarily resistant to or which escaped from tamoxifen treatment. Its preventive activity on tumor take also suggests its use as an adjuvant to prevent the development of metastases.