Synthesis and Evaluation of Pyrimidine Steroids as Antiproliferative Agents.

A small and focused library of steroidal non-fused and fused pyrimidines was prepared from pregnenolone acetate and diosgenin, respectively. The key step was the cycloaddition reaction of nitrogen-containing 1,3-binucleophiles with the steroidal α,ß-unsaturated ketone. Urea, thiourea and guanidine reacted in a similar manner and afforded the steroidal pyrimidines in good yields. The antiproliferative tests against human tumor cell lines gave GI50 values in the micromolar range and had no effect on healthy fibroblasts. Additional experiments indicated that the compounds did not act as P-glycoprotein substrates, thus avoiding the rise of drug resistance. The fused steroidal pyrimidinethione was selected as drug lead for further testing due to its strong antiproliferative activities within the low micromolar range.

Cortisol, progesterone, 17αOHprogesterone, and pregnenolone in foals born from mare's hormone-treated for experimentally induced ascending placentitis.

This study aimed to evaluate steroid hormones in foals born from mares treated for ascending placentitis with different combinations of trimethoprim-sulfamethoxazole (TMS), flunixin meglumine (FM), long-acting altrenogest (ALT) and estradiol cypionate (ECP) for ten consecutive days, starting two days after experimental induction of placentitis with Streptococcus zooepidemicus. Fourty-six pregnant mares and respective foals were assigned as healthy group (Control, n = 8) or treated groups as follows: TMS+FM (n = 8), TMS+FM+ALT (n = 8), TMS+FM+ALT+ECP (n = 6), TMS+FM+ECP (n = 6) and no treatment (NO TREAT n = 10). At delivery, foals were classified as high-risk or low-risk based on clinical and hematologic findings, and survival rates were recorded during the first week of life for comparisons across groups. Cortisol, progesterone, 17αOHprogesterone, and pregnenolone concentrations were determined via immunoassays in 31 of the 46 foals immediately after foaling (0 h), at 12, 24, 48 h, and seven days post-partum (168h). At birth, serum cortisol concentrations were higher in Control and TMS+FM+ECP foals than in remaining groups (p < 0.05). Foals in TMS+FM+ALT and TMS+FM groups had higher 17αOHprogesterone concentrations at 24 h and 48 h, respectively (p < 0.05). Pregnenolone concentrations were higher in TMS+FM than TMS+FM+ALT+ECP foals at 7 days (p < 0.05). High-risk and non-surviving foals had decreased concentrations of cortisol at parturition, but increased concentrations of progesterone from 0 h to 48 h. Pregnenolone and 17αOHprogesterone concentrations were increased and pregnenolone after 12 h in high-risk and non-surviving foals (p < 0.05). In conclusion, adding ECP to the treatment of experimentally-induced placentitis appears to improve foal viability and endocrine response. Cortisol and progestogen profiles were abnormal in high-risk and non-surviving foals, and those treated with ALT or TMS+FM only.

Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions.

BACKGROUND: Since noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE) in the steroid hormone profile of a serum-free granulosa cell (GC) culture system in the context of follicular development and dominance. METHODS: Primary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs. RESULTS: GCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose-response study. The highest tested concentration of NE (10 (-7) M) resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone production was further investigated by incubating GCs with propranolol (10 (-8) M), a non-selective beta-adrenergic antagonist. CONCLUSIONS: The present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.

Cisplatin inhibits testosterone synthesis by a mechanism that includes the action of reactive oxygen species (ROS) at the level of P450scc.

Cisplatin (Cs) is a chemotherapeutic agent able to generate reactive oxygen species (ROS) which are linked to several side effects of the drug. Even when it is known that Cs produces Leydig cell dysfunction, it is unknown whether this particular side effect is mediated by ROS. The aim of this study was to evaluate the in vitro effects of Cs on testosterone production and the participation of ROS in this effect. We demonstrate that Cs promotes the generation of ROS in a time-, and concentration-dependent fashion, not only in mouse testicular interstitial cells but also in MA-10 Leydig cells. Also, Cs inhibits testosterone synthesis in a concentration-dependent fashion (5-50 µM for 4 h) and to a similar extent, in cells exposed to human chorionic gondadotropin hormone (hCG), to an analog of the second messenger cAMP (8Br-cAMP) or to a freely diffusible cholesterol analog (22R-hydroxycholesterol). However, this treatment does not inhibit the conversion of pregnenolone to testosterone. These data suggest that Cs exerts its inhibitory action on testosterone synthesis by an action at the level of P450scc. We also demonstrated that an antioxidant impairs the inhibitory effect of Cs on the conversion of the cholesterol analog into pregnenolone and that Cs does not change the expression level of P450scc mRNA. Therefore, it is concluded that Cs inhibits testosterone synthesis by a mechanism that includes the inhibition of P450scc by ROS.

Glibenclamide-pregnenolone derivative has greater hypoglycemic effects and biodistribution than glibenclamide-OH in alloxan-rats.

AIM: The present study was designed to investigate the activity of two glibenclamide derivatives on glucose concentration. An additional aim was to identify the biodistribution of glibenclamide derivatives in different organs in a diabetic animal model. METHODS: The effects of two glibenclamide derivatives on glucose concentration were evaluated in a diabetic animal model. In addition, glibenclamide derivatives were bound to Tc-99m using radioimmunoassay methods. To evaluate the pharmacokinetics of the glibenclamide derivatives over time (15, 30, 45 and 60 min) the Tc-99m-glibenclamide conjugates were used. RESULTS: The results showed that glibenclamide-pregnenolone had greater hypoglycemic activity than glibenclamide or glibenclamide-OH. The data also showed that the biodistribution of Tc-99m-glibenclamide-OH in all organs was less than that of the Tc-99m-glibenclamide-pregnenolone derivative. CONCLUSIONS: The glibenclamide-pregnenolone derivative had greater hypoglycemic effects and its biodistribution was wider than glibenclamide-OH. The data suggest that the steroid nucleus may be important to the hypoglycemic activity of the glibenclamide-pregnenolone derivative and this could be related to the degree of lipophilicity induced by the steroid nucleus in the chemical structure of glibenclamide-pregnenolone.

Localization and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1) in the adult rat kidney.

Cumulative evidence demonstrated effective downstream metabolism of pregnenolone in renal tissue. The aim of this study was to evaluate the expression and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1), which converts cholesterol into pregnenolone, in adult rat kidney. Immunohistochemical labeling for CYP11A1 was observed in renal cortex and medulla, on structures identified as distal convoluted tubule and thick ascending limb of Henle's loop, respectively. Immunoblotting analysis corroborated the renal expression of the protein in inner mitochondrial membrane fractions. The incubation of isolated mitochondria with the membrane-permeant cholesterol analogue 22R-hydroxycholesterol resulted in efficient formation of pregnenolone, the immediate precursor for the synthesis of all the steroid hormones. The low progesterone production rate observed in these experiments suggested a poor activity of 3ß-hydroxysteroid dehydrogenase enzyme in renal mitochondria. The steroidogenic acute regulatory protein (StAR), involved in the mitochondrial import of cholesterol, was detected in renal tissue at both mRNA and protein level. Immunostaining for StAR showed similar distribution to that observed for CYP11A1. The expression of StAR and CYP11A1 was found to be higher in medulla than in cortex. This enhanced expression of steroidogenesis-related proteins correlated with a greater pregnenolone synthesis rate and higher steroid hormones tissular content measured in medulla. In conclusion, we have established the expression and localization of StAR and CYP11A1 protein, the ability of synthesizing pregnenolone and a region-specific content of sex hormones in the adult rat kidney. These data clearly show that the kidney is a steroid hormones synthesizing organ. It is proposed that the existence in the kidney of complete steroidogenic machinery would respond to a physiological significance.

Pregnenolone sulfate infused in lateral septum of male rats impairs novel object recognition memory.

In the present paper we show for the first time that pregnenolone sulfate (Preg-S) impairs rats' memory for novel object recognition when injected in lateral septum (1.2 microM). The effect of Preg-S is clearly related to the moment the reagent is administered: if administered shortly after the training phase, or prior to the test phase of the experiment, there is no amnesic effect. It is only amnesic when administered 30 min before training. Accordingly, Preg-S does not appear to affect the storage of new memories or their retrieval but rather the acquisition itself. Based on the described afferences and efferences of lateral septum, we suggest a possible stimulatory effect of Preg-S regarding glutamate receptors and/or an inhibitory effect of GABA receptors located in local interneurons or recurrent axon collaterals, both of which have been reported to exist in the aforementioned nucleus.

Effects of two new steroids and cyproterone on some biomarkers of oxidative stress and serotonergic system on rat prostate and brain.

The purpose of this study was to evaluate the effect of cyproterone acetate (CPA, A) compared with new synthetic steroids 3alpha-acetoxy-5,6-epoxy-16-pregnen-20-one (B) and 17alpha-hydroxy-16beta-methyl-1,4,6-pregnatriene-3,20-dione (C) in rat prostate and brain. Groups of animals were treated either with A, B or C (4 mg kg(-1) day(-1)) by the intraperitoneal route for 5 days. Levels of reduced glutathione (GSH), 5-hydroxy-indole acetic acid (5-HIAA), lipid peroxidation (as thiobarbituric acid reactive substances, TBARS) and the activities of Na(+), K(+)- and total ATPases were assayed in prostate and brain for each group of animals including a control group. No appreciable changes were shown in Na(+), K(+)-ATPase and total ATPases and TBARS on prostate and brain of rats that received A, B and C steroids. However, the levels of GSH and 5-HIAA decreased significantly (P < 0.05) in both tissues for the steroids assayed. It is concluded that CPA and the homologues B and C steroids induce changes in the levels of GSH and serotonin in rat prostate and brain.

[Activity induced by androsterone and hemisuccinate of androsterone on perfusion pressure and vascular resistance]. / Actividad inducida por androsterona y hemisuccinato de androsterona sobre la presión de perfusión y la resistencia vascular.

INTRODUCTION: Few data exist with respect to the effects of androsterone and their derivatives at cardiovascular level. In addition, the molecular mechanisms and cellular site of action of these androgens are still unclear. OBJECTIVE: An evaluation was conducted on the effects induced by androsterone and hemisuccinate of androsterone on perfusion pressure and vascular resistance. MATERIALS AND METHODS: The effects of both androsterone and hemisuccinate of androsterone on the perfusion pressure and vascular resistance in isolated rat hearts (Langendorff model) were evaluated. RESULTS: The results showed that: (1) the hemisuccinate of androsterone [10(-9) M] increases the perfusion pressure and vascular resistance in comparison with the androsterone [10(-9) M]; (2) the effect of androsterone-derivative [10(-9) M-10(-5) M] on perfusion pressure not was inhibited by indometacin [10(-6) M]; (3) nifedipine [10(-6) M] blocks the effects exerted by hemisuccinate of androsterone [10(-9) M-10(-5) M] on perfusion pressure; and (4) the effect of androsterone-derivative [10(-9) M-10(-5) M] on perfusion pressure in presence of flutamide [10(-6) M] was inhibited. CONCLUSIONS: The effects induced by androsterone and hemisuccinate of androsterone on the perfusion pressure and resistance vascular probably involve the interaction of steroid-receptor androgenic and, indirectly, activation of the calcium channel to induce variations in the perfusion pressure.

In vivo and in vitro effect of novel 4,16-pregnadiene-6,20-dione derivatives, as 5alpha-reductase inhibitors.

In this study, we report the synthesis and biological evaluation of several new 3-substituted pregna-4,16-diene-6,20-dione derivatives (11a-11d). These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological effect of these steroids was demonstrated in in vivo and in vitro experiments. In the in vivo experiments, we measured the activity of the 11a-11d on the weight of the prostate gland of gonadectomized hamsters treated with testosterone plus finasteride or with the new steroids. For the studies in vitro, we determined the IC50 values by measuring the steroid concentration that inhibits 50% of the activity of 5alpha-reductase present in human prostate. In order to study the mechanism of action of 11a-11d, we also determined the capacity of these steroids to bind to the androgen receptor (AR) present in the rat prostate cytosol using labeled mibolerone as a tracer. The results from this work indicated that compounds 11a-11d significantly decreased the weight of the prostate as compared to testosterone treated animals and this reduction of the weight of the prostate was comparable to that produced by the finasteride. On the other hand 11a-11d exhibited a high inhibitory activity for the human 5alpha-reductase enzyme with IC50 values of 1.4 x 10(-8), 1.8 x 10(-9), 1.0 x 10(-8) and 4 x 10(-5) respectively. However the IC50 value of 11a (1.8 x 10(-9)) was the only one lower than that of finasteride (8.5 x 10(-9)). Nevertheless this compound did not show a higher potency in vivo as compared to that of compounds 11b-11d. The competition analysis for the androgen receptor indicated that the IC50 value of non-labeled mibolerone used in this experiment was 1nM, whereas steroids 10, 11a-11d did not inhibit the labeled mibolerone binding to the androgen receptor. On the other hand, steroid 10 did not show any activities in vitro or in vivo, and for this reason these steroidal derivatives (11a-11d) cannot be considered as prodrugs of compound 10. In conclusion, the compounds containing chlorine 11a, bromine 11b, iodine 11c atoms, and 11d (without any substituent in the ester moiety) at C-3 produce a significant decrease of the prostate weight in castrated animals treated with T and inhibits the activity of the 5alpha-reductase. Apparently the presence of the halogen atoms in compounds 11a-11c enhances the inhibitory activity for the 5alpha-reductase enzyme.

The key steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase in Taenia solium and Taenia crassiceps (WFU).

Larval and adult stages of Taenia solium and Taenia crassiceps WFU strain were analyzed by histochemical and biochemical methods to determine the existence of steroid pathways. The presence of the key enzyme 3beta-hydroxisteroid-dehydrogenase (3beta-HSD) was examined in frozen sections of cysticerci obtained from mice and segments of tapeworms obtained from the intestine of hamsters. 3beta-HSD activity was detected by nitroblue-tetrazolium products after incubation with dehydroepiandrosterone, androstendiol, or pregnenolone. Tapeworm tissues exhibited 3beta-HSD activity in the subtegumentary areas of the neck and immature proglottids following incubation with androstendiol, as well as surrounding the testes in mature proglottids. T. solium cysticerci exhibited 3beta-HSD activity in the subtegumentary tissues. The synthesis of steroid hormones involving the activity of 3beta-HSD was studied in cysticerci or tapeworms incubated in the presence of tritiated steroid precursors. The culture media were analyzed by thin layer chromatography and showed synthesis of androstendiol, testosterone, and 17beta-estradiol by cysticerci, androstendiol, and 17beta-estradiol by tapeworms. The results strongly suggest the activity of 3beta-HSD in taeniid parasites that have at least a part of the enzymatic chain required for androgen and estrogen synthesis and that the enzymes are present in the larval stage and from the early strobilar stages to the mature proglottids.

Influence of epipregnanolone on the modulation of rapid tolerance to ethanol by neurosteroids

OBJECTIVE: The objective of the present study was to investigate the effect of epipregnanolone on the influence of neurosteroids on the development of rapid tolerance to the motor impairing and hypothermic effects of ethanol. METHOD: Experiment 1: on Day 1 groups of mice were pretreated with saline or with epipregnanolone. After 30 min each group was further divided in subgroups that received ethanol or saline. Thirty, 60 and 90 min after the injections the animals were tested on the rota-rod or the body temperature was measured. On Day 2 all groups received ethanol and a similar procedure was followed to evaluate rapid tolerance. Experiment 2 and 3: On Day 1 groups of mice were treated with epipregnanolone and after 15 min each group was divided into three groups in order to receive pregnenolone sulfate, dehydroepiandrosterone sulfate or saline. Thirty minutes later, each group was further divided into two subgroups in order to receive ethanol or saline, respectively, and 30, 60 and 90 min later the animals were tested as in the experiment 1. On Day 2 all groups received ethanol and a similar procedure was followed to evaluate rapid tolerance. RESULTS: Pretreatment with epipregnanolone (0.10-0.30 mg/kg) significantly blocked the development of tolerance to the motor impairing and hypothermic effects induced by ethanol in mice. Considering tolerance to ethanol-induced motor impairment, epipregnanolone (0.15 mg/kg) reversed the stimulatory action of dehydroepiandrosterone sulfate (0.15 mg/kg), but did not affect the actions of pregnenolone sulfate (0.08 mg/kg). Moreover, epipregnanolone prevented the inhibitory action of allotetrahydrodeoxycorticosterone (0.10 mg/kg). In relation to ethanol-induced hypothermia, the results showed that pretreatment with epipregnanolone (0.30 mg/kg) significantly prevented the stimulatory action of dehydroepiandrosterone sulfate and pregnenolone sulfate, as well as the inhibitory action of...

OBJETIVO: O objetivo do presente estudo foi o de investigar o efeito da epipregnanolona sobre a influência de neuroesteróides no desenvolvimento da tolerância rápida aos efeitos de incoordenação motora e hipotermia induzidos pelo etanol. MÉTODO: Experimento 1: no Dia 1, grupos de camundongos foram pré-tratados com salina ou com epipregnanolona. Após 30 min, cada grupo foi subdividido recebendo etanol ou salina. Aos 30, 60 e 90 min após as injeções, os animais foram testados no rota-rod ou a temperatura corporal foi avaliada. No Dia 2, todos os grupos receberam etanol e um procedimento similar foi seguido para avaliar a tolerância rápida. O pré-tratamento com a epipregnanolona (0,10-0,30 mg/kg) bloqueou significantemente o desenvolvimento da tolerância aos efeitos de incoordenação motora e hipotermia induzidos pelo etanol em camundongos. Experimento 2 e 3: no Dia 1, grupos de animais foram tratados com epipregnanolona e, após 15 min, cada grupo foi dividido em três grupos para receber sulfato de pregnanolona, sulfato de dehidroepiandrosterona ou salina. Após 30 min, cada grupo foi dividido em dois subgrupos para receber etanol ou salina, respectivamente, e após 30, 60 e 90 min os animais foram testados como no experimento 1. No Dia 2, todos os grupos receberam etanol e 30 min após foram testados como mencionado no experimento 1. RESULTADOS: Considerando a tolerância ao prejuízo motor induzido pelo etanol, a epipregnanolona (0,15 mg/kg) bloqueou a ação estimulatória do sulfato de dehidroepiandrosterona (0,15 mg/kg), mas não afetou a ação do sulfato de pregnanolona (0,08 mg/kg). Entretanto, a epipregnanolona bloqueou a ação inibitória da alotetrahidrodeoxicorticosterona (0,10 mg/kg). Em relação à hipotermia induzida pelo etanol, os resultados demonstraram que o pré-tratamento com epipregnanolona (0,30 mg/kg) bloqueou significantemente a ação estimulatória do sulfato de dehidroepiandrosterona e do sulfato de pregnanolona, bem como a ação...

Influence of epipregnanolone on the modulation of rapid tolerance to ethanol by neurosteroids.

OBJECTIVE: The objective of the present study was to investigate the effect of epipregnanolone on the influence of neurosteroids on the development of rapid tolerance to the motor impairing and hypothermic effects of ethanol. METHOD: Experiment 1: on Day 1 groups of mice were pretreated with saline or with epipregnanolone. After 30 min each group was further divided in subgroups that received ethanol or saline. Thirty, 60 and 90 min after the injections the animals were tested on the rota-rod or the body temperature was measured. On Day 2 all groups received ethanol and a similar procedure was followed to evaluate rapid tolerance. Experiment 2 and 3: On Day 1 groups of mice were treated with epipregnanolone and after 15 min each group was divided into three groups in order to receive pregnenolone sulfate, dehydroepiandrosterone sulfate or saline. Thirty minutes later, each group was further divided into two subgroups in order to receive ethanol or saline, respectively, and 30, 60 and 90 min later the animals were tested as in the experiment 1. On Day 2 all groups received ethanol and a similar procedure was followed to evaluate rapid tolerance. RESULTS: Pretreatment with epipregnanolone (0.10-0.30 mg/kg) significantly blocked the development of tolerance to the motor impairing and hypothermic effects induced by ethanol in mice. Considering tolerance to ethanol-induced motor impairment, epipregnanolone (0.15 mg/kg) reversed the stimulatory action of dehydroepiandrosterone sulfate (0.15 mg/kg), but did not affect the actions of pregnenolone sulfate (0.08 mg/kg). Moreover, epipregnanolone prevented the inhibitory action of allotetrahydrodeoxycorticosterone (0.10 mg/kg). In relation to ethanol-induced hypothermia, the results showed that pretreatment with epipregnanolone (0.30 mg/kg) significantly prevented the stimulatory action of dehydroepiandrosterone sulfate and pregnenolone sulfate, as well as the inhibitory action of allotetrahydrodeoxicorticosterone (0.20 mg/kg), on tolerance to this effect. CONCLUSIONS: The results suggest a differential interaction between neurosteroids that might modulate the development of rapid tolerance to ethanol.

Effect of isopregnanolone on rapid tolerance to the anxiolytic effect of ethanol.

OBJECTIVE: It has been shown that neurosteroids can either block or stimulate the development of chronic and rapid tolerance to the incoordination and hypothermia caused by ethanol consumption. The aim of the present study was to investigate the influence of isopregnanolone on the development of rapid tolerance to the anxiolytic effect of ethanol in mice. METHOD: Male Swiss mice were pretreated with isopregnanolone (0.05, 0.10 or 0.20 mg/kg) 30 min before administration of ethanol (1.5 g/kg). Twenty-four hours later, all animals we tested using the plus-maze apparatus. The first experiment defined the doses of ethanol that did or did not induce rapid tolerance to the anxiolytic effect of ethanol. In the second, the influence of pretreatment of mice with isopregnanolone (0.05, 0.10 or 0.20 mg/kg) on rapid tolerance to ethanol (1.5 g/kg) was studied. CONCLUSIONS: The results show that pretreatment with isopregnanolone interfered with the development of rapid tolerance to the anxiolytic effect of ethanol.

Effect of isopregnanolone on rapid tolerance to the anxiolytic effect of ethanol / Influência da isopregnenolona na tolerância rápida ao efeito ansiolítico do etanol

OBJETIVE: It has been shown that neurosteroids can either block or stimulate the development of chronic and rapid tolerance to the incoordination and hypothermia caused by ethanol consumption. The aim of the present study was to investigate the influence of isopregnanolone on the development of rapid tolerance to the anxiolytic effect of ethanol in mice. METHOD: Male Swiss mice were pretreated with isopregnanolone (0.05, 0.10 or 0.20 mg/kg) 30 min before administration of ethanol (1.5 g/kg). Twenty-four hours later, all animals we tested using the plus-maze apparatus. The first experiment defined the doses of ethanol that did or did not induce rapid tolerance to the anxiolytic effect of ethanol. In the second, the influence of pretreatment of mice with isopregnanolone (0.05, 0.10 or 0.20 mg/kg) on rapid tolerance to ethanol (1.5 g/kg) was studied. CONCLUSIONS: The results show that pretreatment with isopregnanolone interfered with the development of rapid tolerance to the anxiolytic effect of ethanol.

OBJETIVO: Estudos prévios têm mostrado que os neuroesteróides podem bloquear ou estimular o desenvolvimento da tolerância rápida e crônica aos efeitos de incoordenação e hipotermia produzidos pelo etanol. O objetivo do presente estudo foi investigar a influência da isopregnenolona sobre o desenvolvimento da tolerância rápida ao efeito ansiolítico do etanol em camundongos. MÉTODO: Camundongos suíços, machos, foram pré-tratados com isopregnenolona (0,05, 0,10 ou 0,20 mg/kg) 30 minutos antes da administração de etanol (1,5 g/kg). Após 24 horas, todos os animais foram testados no labirinto em cruz elevado. O primeiro experimento foi realizado com o intuito de selecionar uma dose de etanol que produzisse tolerância rápida ao efeito ansiolítico do etanol. No segundo experimento, o objetivo foi investigar o efeito da isopregnenolona (ISO; 0,05, 0,10 ou 0,20 mg/kg) sobre a tolerância rápida ao etanol (1,5 g/kg). CONCLUSÕES: Os resultados mostram que o tratamento prévio com isopregnenolona interferiu no desenvolvimento da tolerância rápida ao efeito ansiolítico etanol.

Effect of neurosteroids on the retinal gabaergic system and electroretinographic activity in the golden hamster.

It has been established that neurosteroids can either inhibit or enhance GABA(A) receptor activity. Although GABA is the main inhibitory neurotransmitter in the mammalian retina, the effects of neurosteroids on retinal GABAergic activity have not been investigated. The aim of this work was to study the neurochemical and electroretinographic effects of neurosteroids in the golden hamster. On one hand, pregnenolone sulfate inhibited and allotetrahydrodeoxycorticosterone increased GABA-induced [36Cl]- uptake in neurosynaptosomes. On the other hand, in whole retinas, pregnenolone sulfate increased, whereas allotetrahydrodeoxycorticosterone decreased high potassium-induced [3H]GABA release. The effect of both neurosteroids on GABA release was Ca2+-dependent, as in its absence release was not altered. The intravitreal injection of pregnenolone sulfate or vigabatrin (an irreversible inhibitor of GABA degradation) significantly decreased scotopic b-wave amplitude, whereas the opposite effect was evident when bicuculline or allotetrahydrodeoxycorticosterone were injected. A protein with a molecular weight close to that of hamster adrenal cytochrome P450 side-chain cleavage (P450scc) was detected in the hamster retina. P450scc-like immunoreactivity was localized in the inner nuclear and the ganglion cell layers. These results indicate that neurosteroids significantly modulate retinal GABAergic neurotransmission and electroretinographic activity. In addition, the selective localization of P450scc suggests that neurosteroid biosynthesis might occur only in some layers of the hamster retina.

New 5alpha-reductase inhibitors: in vitro and in vivo effects.

The enzyme 5alpha-reductase is responsible for the conversion of testosterone (T) to its more potent androgen dihydrotestosterone (DHT). This steroid had been implicated in androgen-dependent diseases such as: benign prostatic hyperplasia, prostate cancer, acne and androgenic alopecia. The inhibition of 5alpha-reductase enzyme offers a potentially useful treatment for these diseases. In this study, we report the synthesis and pharmacological evaluation of several new 3-substituted pregna-4, 16-diene-6, 20-dione derivatives. These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological activity of the new steroidal derivatives was determined in vivo as well as in vitro experiments. In vivo experiments, the anti-androgenic effect of the steroids was demonstrated by the decrease of the weight of the prostate gland of gonadectomized hamster treated with T plus finasteride or the new steroids. The IC50 value of these steroids was determined by measuring the conversion of radio labeled T to DHT. The results of this study carried out with 5alpha-reductase enzyme from hamster and human prostate showed that four of the six steroidal derivatives (5, 7, 9, 10) exhibited much higher 5alpha-reductase inhibitory activity, as indicated by the IC50 values than the presently used Proscar 3 (finasteride). The comparison of the weight of the hamster's prostate gland indicated that compound 5 had a comparable weight decrease as finasteride. The overall data of this study showed very clearly those compounds 5, 7, 9, 10 are good inhibitors for the 5alpha-reductase enzyme.

Adrenocorticotropin induces heme oxygenase-1 expression in adrenal cells.

Heme oxygenase (HO) catalyzes the first and rate-controlling step of heme catabolism into biliverdin, iron and carbon monoxide. Three isoforms of HO have been identified so far: the inducible HO-1 and the constitutive HO-2 and HO-3. Both HO-1 and HO-2 were expressed in zona fasciculata (ZF) adrenal cells and in a mouse adrenocortical cell line (Y1). HO-1 but not HO-2 expression was upregulated by adrenocorticotropic hormone (ACTH) and accumulation of HO-1 protein correlated with an increase in HO activity in Y1 cells. ACTH induced HO-1 expression in a time- and dose-dependent manner with a maximum after 5 h of treatment and a threshold concentration of 0.1 mIU/ml. Actinomycin D and cycloheximide completely blocked the effect of ACTH on HO-1 mRNA expression whereas mRNA stability was not affected by ACTH. Permeable analogs of cAMP mimicked the effect of ACTH on HO-1 expression and ACTH induction was prevented by the protein kinase A (PKA) inhibitor H89. Steroid production was significantly increased when both HO-1 and HO-2 activities were inhibited by Sn-protoporphyrin IX (SnPPIX). The lipid peroxidation and increase in carbonyl content triggered by hydrogen peroxide was prevented by treatment of Y1 cells with bilirubin and ACTH.

Two prevalent CYP17 mutations and genotype-phenotype correlations in 24 Brazilian patients with 17-hydroxylase deficiency.

We performed molecular genetic analysis of 24 subjects from 19 families with 17-hydroxylase deficiency in Brazil. Of 7 novel CYP17 mutations, 2 (W406R and R362C) account for 50% and 32% of the mutant alleles, respectively. Both mutations were completely inactive when studied in COS-7 cells and yeast microsomes; however, phenotypic features varied among subjects. Some 46,XY individuals with these genotypes had ambiguous genitalia, and other subjects had normal blood pressure and/or serum potassium. We found mutations W406R and R362C principally in families with Spanish and Portuguese ancestry, respectively, suggesting that two independent founder effects contribute to the increased prevalence of 17-hydroxylase deficiency in Brazil. Mutations Y329D and P428L retained a trace of activity, yet the two individuals with these mutations had severe hypertension and hypokalemia. The 46,XX female with mutation Y329D reached Tanner stage 5, whereas the 46,XY subject with mutation P428L remained sexually infantile. The severity of hypertension, hypokalemia, 17-deoxysteroid excess, and sex steroid deficiency varied, even among patients with completely inactive CYP17 protein(s). Spontaneous sexual development occurred only in 46,XX females with partial deficiencies. We conclude that other factors, in addition to CYP17 genotype, contribute to the phenotype of individual patients with 17-hydroxylase deficiency.

Steroid structural requirements for stabilizing or disrupting lipid domains.

In artificial membrane bilayers, saturated long acyl chain-containing phospholipids and cholesterol (Chol) interact to form more ordered domains than those in phospholipids with unsaturated or short fatty acyl chains. We have extended the fluorescence techniques of London et al. [Xu, X., and London, E. (2000) Biochemistry 39, 843-849; Xu, X., Bittman, R., Duportail, G., Heissler, D., Vilchezes, C., and London, E. (2001) J. Biol. Chem. 276, 33540-33546] to study the propensity of several steroids to form or disrupt such ordered lipid domains. Temperature-dependent fluorescence quenching and steady-state polarization of the extrinsic fluorescent probe diphenylhexatriene (DPH) in model membranes composed of dipalmitoylphosphatidylcholine (or sphingomyelin), a nitroxide spin-labeled phosphatidylcholine (12-SLPC), and a given steroid were combined to study the influence of the latter on (a) ordered lipid domain formation, (b) stabilization, and (c) the extension of the ordered lipid assemblies. The results of the two totally independent methods, fluorescence quenching by 12-SLPC and fluorescence polarization of DPH, show that all steroids examined, except for Chol and 25-hydroycholesterol, behave as lipid domain-disrupting compounds. Additionally, we found a positive correlation between the hydrophobicity of steroids and their ordered lipid domain-promoting activity. Comparison of the chemical structures disclosed some distinctive traits of ordered lipid domain-promoting steroids: (i) the presence of an isooctyl side chain bond at C17; (ii) the absence of carbons attached to C23 (i.e., C24-C27) in any of the other (domain-disrupting) steroids; (iii) the presence of a small polar group at position C3; and (iv) the absence of polar groups in the fused rings, with the exception of substitutions at position C3 in the A ring.