Glycine soya diet synergistically enhances the suppressive effect of tamoxifen and inhibits tamoxifen-promoted hepatocarcinogenesis in 7,12-dimethylbenz[α]anthracene-induced rat mammary tumor model

There is increasing interest in phytoestrogens as potential alternatives to synthetic selective estrogen receptor modulators (SERMs) in the prevention and therapy of breast cancer. The present study is aimed at determining whether dietary glycine soya (Glycine max seeds; GS), which is rich in phytoestrogens, can enhance the anti breast cancer efficacy of the SERM tamoxifen (TAM) and the effect of TAM and GS, either alone or in combination, on DMBA-initiated hepatocarcinogenesis in rat. For determination of enhancing effect, rats bearing palpable 7, 12-dimethylbenz[α] anthracene (DMBA)-induced mammary tumors were treated with TAM (10 mg kg⁻¹/day) while being fed AIN-93G diet with or without added GS (3 × 10⁴ mg kg⁻¹), and the tumor growth was monitored up to 5 weeks of treatment. For determining the effect on hepatocarcinogenesis, DMBA-initiated rats were exposed to TAM and dietary GS as above for 6 weeks during promotion stage in a medium-term bioassay, and the development of placental form of glutathione-S-transferase (GST-P)-expressing preneoplastic liver lesions was quantified. Exposure to both TAM and dietary GS enhanced the anti tumor efficacy of TAM via a combination of tumor cell apoptosis (determined by TUNEL) and inhibition of tumor cell proliferation (determined by PCNA immunostaining) and suppressed the growth of GST-P-positive liver lesions. The findings show that dietary GS enhances the therapeutic efficacy of TAM against mammary tumors and minimizes TAM’s hepatocarcinogenesis promotion potential.     

Therapeutic effect of centchroman alone and in combination with glycine soya on 7,12-dimethylbenz[α]anthracene-induced breast tumor in rat

Centchroman is a non-steroidal oral contraceptive and has been found to be a candidate drug for breast cancer exhibiting partial to complete remission of lesions in 40.5% of breast cancer patients. The therapeutic efficacy of centchroman was monitored alone and together with glycine soya on growth of 7,12-dimethylbenz[α]anthracene-induced breast tumor in rat. The tumor regression was monitored at different doses of centchroman alone ranging from 0 to 10 mg kg⁻¹ and with glycine soya from 1 × 10⁴ to 5 × 10⁴ mg kg⁻¹ per day until 5 weeks treatment. An optimum tumor treatment opus was established with varying treatment parameters including doses of therapeutic agents and treatment period. The tumors were found to be static with a strong anti-estrogenic effect. Overall our study shows that both centchroman and glycine soya alone and jointly combat with breast cancer.     

Evaluation of the effects of ellagic acid (EA) on 7,12-dimethylbenz(α) anthracene (DMBA) induced micronuclei in mammalian cells in vitro and in vivo

We evaluated the protective effects of EA, a promising dietary constituent against degenerative diseases, on the clastogenic action of the model carcinogen DMBA in vitro on human hepatoma cells (HepG2) and in vivo on bone marrow of mice, using the frequencies of induced micronuclei as the end point. Pre-, post- and simultaneous treatments with EA and the carcinogen were carried out in vitro. Simultaneous treatment with EA caused a statistically significant increase of DMBA induced MN, suggesting a direct interaction between the two agents. No significant reduction in DMBA induced MN was found by pre- or post treatment with EA. Similar effects were observed in the toxicity assay. In in vivo experiments, EA pre-treatment did not affect the frequencies of MN in PCEs of bone marrow induced by DMBA. A good correlation was found between in vitro and in vivo experiments. Our results did not reveal any clear indication on the efficacy of EA on the induction of micronuclei by DMBA. EA by itself did not show any harmful effects.     

Role of pregnane xenobiotic receptor in the midbrain ventral tegmental area for estradiol- and 3α,5α-THP-facilitated lordosis of female rats

Rationale

Progesterone and its metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP), have actions in the ventral tegmental area (VTA) that are required for lordosis, a characteristic mating posture of female rodents. 17β-estradiol (estradiol) co-varies with progestogens over natural cycles, enhances production of 3α,5α-THP, and is required for successful reproductive behavior.

Objectives

A question of interest is the role of pregnane xenobiotic receptor (PXR), a nuclear receptor that regulates enzymes needed for the production of 3α,5α-THP, for estradiol-mediated lordosis. The hypothesis tested was that if PXR is involved in estradiol-mediated biosynthesis of 3α,5α-THP and reproductive behavior, knocking down expression of PXR in the VTA of estradiol-primed, but not vehicle-primed, rats should decrease lordosis and midbrain 3α,5α-THP; effects may be attenuated by 3α,5α-THP administered to the VTA.

Methods

Ovariectomized rats were administered subcutaneous injections of oil vehicle or estradiol. Rats were then administered PXR antisense oligonucleotides (PXR AS-ODNs; which are expected to locally knock down expression of PXR), or control (saline), infusions to the VTA. Rats were administered 3α,5α-THP or vehicle via infusions to the VTA. Reproductive behavior (paced mating task) of rats was determined in addition to exploratory (open field), affective (elevated plus maze), and pro-social (social interaction task) behavior.

Results

Reproductive behavior (i.e., increased lordosis) was enhanced with estradiol-priming and infusions of 3α,5α-THP to the VTA. Infusions of PXR AS-ODNs to the VTA attenuated responses in estradiol-, but not vehicle-, primed rats, compared to control infusions.

Conclusions

PXR may be involved in a neuroregulatory response involving biosynthesis of 3α,5α-THP in the midbrain VTA of estradiol-primed rats.     

A comparative study of the anticlastogenic effects of chlorophyllin on N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or 7,12-dimethylbenz (α) anthracene (DMBA) induced micronuclei in mammalian cells in vitro and in vivo

Chlorophyllin (CHL), a water soluble derivative of chlorophyll has been shown to have both anticarcinogenic and antigenotoxic properties. We evaluated the protective effects of CHL (25 μM in vitro, 4 and 100 mg/kg. b.w.) on the clastogenic action of two model carcinogens, MNNG and DMBA (25 μM and 2 μM respectively) in vitro on human hepatoma cells (HepG2) and (40 mg and 25 mg/Kg/b.w. respectively) in vivo on bone marrow of mice, using the frequencies of induced micronuclei as the end point. Pre-, post- and simultaneous treatments with CHL and the carcinogen were carried out in vitro. With MNNG, only simultaneous treatment with CHL was effective in reducing the frequencies of MN, suggesting a direct interaction between CHL and MNNG. A statistically significant reduction in of DMBA induced MN was found by pre-or post treatment with CHL while a reduction (not significant) was observed by simultaneous treatment. In in vivo experiments, CHL pre-treatment did not affect the frequencies of MN in PCEs of bone marrow induced by MNNG or DMBA. However, increased the toxic effect of DMBA (reduction in percent of PCEs) was accompanied by a reduction in the induced frequencies of MN. CHL was not clastogenic in both in vitro and in vivo tests. It can be concluded that (a) CHL has a protective effect against MNNG and DMBA. This effect is dependent upon the protocol employed in in vitro experiments. In vivo, CHL did not have a protective effect against MNNG and DMBA. A protective effect of CHL against DMBA was evident only at high toxic levels.     

Metabolism of α-olefin sulfonate (AOS) in rats

The metabolic fate of ¹⁴C-AOS (a mixture of ¹⁴C-sodium alkenyl(2) sulfonate and ¹⁴C-sodium 3-hydroxy alkane sulfonate) has been studied in rats by a single oral and intravenous injection of 100 mg (50 μCi)/kg and 10 mg (5 μCi)/kg, respectively. After oral administration, ¹⁴C-AOS was rapidly absorbed from the gastrointestinal tract. The blood level of ¹⁴C-activity reached its peak 3 hr after dosing and then declined. Twenty-four hr after the dose, about 0.8%/g of the ¹⁴C-AOS given was detected in cecum content, but in other tissues the figures were under 0.02% dose/g. Within 24 hr after the dose, 72% of the dose was excreted in the urine and 22% in the feces, while the excretion in the bile was 4.3% within 12 hr. The administered radioactivity was rapidly eliminated from the whole body within 24 hr. After intravenous injection, half of the administered dose of radioactivity was excreted within 1 hr. In the 0–6 hr interval post-dose, 90% of the dose was excreted in the urine. No intact ¹⁴C-AOS was detected in any of the urine samples after oral and intravenous doses. The metabolite was apparently more polar than intact ¹⁴C-AOS, and results from data of electrophoresis and equilibrium dialysis indicated that infact ¹⁴C-AOS can bind with proteins, while the metabolites cannot. The metabolite was found to contain alcoholic, unsaturated and sulfonic acid functionalities. It is suggested that the metabolite may be a hydroxylated or polyhydroxylated sulfonic acid of shorter chain length than AOS. These results suggest that ¹⁴C-AOS is rapidly absorbed, metabolized and excreted, therefore, no accumulation of ¹⁴C-AOS occurs.     

Acute inhalation toxicity of aliphatic (C1–C5) nitrites in rats

The 4-hr inhalation LC50 was determined for methyl-, ethyl-, n-propyl-, n-butyl-, isobutyl-, and isopentyl nitrite in Sprague-Dawley rats. LC50 values were 176, 160, 300, 420, 777, and 716 ppm, respectively. The dose-mortality curves were characterized by extremely steep slopes. Toxic signs observed during exposure included cyanosis, prostration, and rarely, convulsions. There were no effects of exposure on body weight gain during a 14-day postexposure observation period. Signs of pulmonary hemorrhage were apparent in rats which died during exposure but were much less prominent in rats sacrificed at study termination. No animals died after cessation of exposure, and rapid recovery was apparent after exposure. Concentration × Time (CT) relationships suggested that the actual concentration was more important than the “dose” in determining the lethal effects of inhalation exposure to nitrites. Because of the extremely steep dosemortality curves, the aliphatic nitrites are more hazardous than the LC50 values would indicate.     

Role of phospholipase D in regulation of testicular Leydig cell hyperplasia in Sprague–Dawley rats treated with di(2-ethylhexyl) phthalate

This study was conducted to determine the functional role of phospholipase D (PLD) involved in testicular Leydig cell damage caused by di (2-ethylhexyl) phthalate (DEHP) in Sprague–Dawley rats. DEHP (500 mg/kg/day) was administered orally to prepubertal rats for 1, 7, 14, 21 or 28 days. After 7 days of exposure, DEHP produced morphological changes in the testis, including alterations in seminiferous tubule diameters and loss of spermatogenic cells. Immunohistochemistry (IHC) analyses revealed that DEHP increased Leydig cell number in the testes as well as significantly increased the expression of PLD1/2 in Leydig cells after 7 days of exposure. Furthermore, the protein levels of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) increased in a similar manner to the PLD1/2 expression patterns. DEHP significantly reduced the expression of sperm-associated antigen 4 (Spag4) and lactate dehydrogenase A (LDHA) mRNA. In contrast, there was a significant increase in the expression of steroidogenic acute regulatory (StAR) mRNA against DEHP in a time-dependent manner, but serum testosterone concentration was decreased. These findings demonstrate that DEHP induces PLD expression in the testicular Leydig cells; this plays a key role in hyperplasia of Leydig cells and steroidogenic pathway via pERK1/2 activation.     

Ethanol Alters Local Cellular Levels of (3α,5α)-3-Hydroxypregnan-20-one (3α,5α-THP) Independent of the Adrenals in Subcortical Brain Regions

The neuroactive steroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP or allopregnanolone) is a positive modulator of GABA_A receptors synthesized in the brain, adrenal glands, and gonads. In rats, ethanol activates the hypothalamic–pituitary–adrenal axis and elevates 3α,5α-THP in plasma, cerebral cortex, and hippocampus. In vivo, these effects are dependent on both the pituitary and adrenal glands. In vitro, however, ethanol locally increases 3α,5α-THP in hippocampal slices, in the absence of adrenal influence. Therefore, it is not known whether ethanol can change local brain levels of 3α,5α-THP in vivo, independent of the adrenals. To directly address this controversy, we administered ethanol (2 g/kg) or saline to rats that underwent adrenalectomy (ADX) or received sham surgery and performed immunohistochemistry for 3α,5α-THP. In the medial prefrontal cortex (mPFC), ethanol increased 3α,5α-THP after sham surgery, compared with saline controls, with no ethanol-induced change in 3α,5α-THP following ADX. In subcortical regions, 3α,5α-THP was increased independent of adrenals in the CA1 pyramidal cell layer, dentate gyrus polymorphic layer, bed nucleus of the stria terminalis, and paraventricular nucleus of the hypothalamus. Furthermore, ethanol decreased 3α,5α-THP labeling in the nucleus accumbens shore and central nucleus of the amygdala, independent of the adrenal glands. These data indicate that ethanol dynamically regulates local 3α,5α-THP levels in several subcortical regions; however, the adrenal glands contribute to 3α,5α-THP elevations in the mPFC. Using double immunofluorescent labeling we determined that adrenal dependence of 3α,5α-THP induction by ethanol is not due to a lack of colocalization of 3α,5α-THP with the cholesterol transporters steroidogenic acute regulatory protein (StAR) or translocator protein (TSPO).     

Subchronic toxicity evaluation of γ-aminobutyric acid (GABA) in rats

γ-Aminobutyric acid (GABA) is an amino acid compound contained in vegetables such as tomatoes and also widely distributed in mammals. GABA acts as an inhibitory neurotransmitter and promotes parasympathetic activity to provide several beneficial effects, for instance, relaxation, anti-stress, and insomnia. GABA, produced via a fermentation process, has been available as a functional food ingredient. As part of a program to assess its safety, GABA was administered by oral gavage at doses of 500, 1250, and 2500 mg/kg body weight to groups of 10 male and 10 female Sprague–Dawley rats for 13 weeks. Treatment was not associated with the test substance-related mortality and appeared to be well tolerated. There were no toxicologically and statistically significant changes in urinalysis, hematology, clinical chemistry parameters, and in necropsy findings. A few statistically significant changes in food consumption and body weights were noted in the male groups while any significant changes were not noted in female groups. There was no effect of treatment on organ weights or on the results of the histopathological examinations. The results of toxicity evaluation support the safety use of GABA and the potential use as a functional food ingredient.     

cis-trans-Isomerization of [E]-5-(2-bromovinyl)-2,2′-anhydrouridine in vivo in rats

1. [E]-5-(2-bromovinyl)-2,2′-anhydrouridine ([E]BVANUR) has considerable antiviral activity against herpes simplex virus type 1 (HSV-1).

2. [E]BVANUR is not a substrate of pyrimidine nucleoside phosphorylases, but it is an inhibitor of uridine phosphorylase (K_i=450 nM).

3. [E]BVANUR (trans-isomer, parent compound) undergoes isomerization to [Z]BVANUR (cis-isomer), the only metabolite in rat, which was identified by?h.p.l.c., mass spectra and n.m.r. spectroscopy.

4. Absorption of the drug from the gastrointestinal tract after oral administration is minimal. Absorption of [E]BVANUR from the abdominal cavity after i.p. administration was slow.     

Failure of the urethane antagonist—Lysergic acid-diethylamide (LSD-25)—To inhibit lung carcinogenesis or the initiating phase of skin carcinogenesis in mice

The fact that LSD-25 (lysergic acid-diethylamide) has been shown to be a powerful antagonist against the anaesthetic and other neurological effects of urethane, raised the question of whether it would also antagonize its carcinogenic action. The present investigation failed to elicit any antagonism by LSD-25 against the carcinogenic action on the lung, or against the initiating phase of skin carcinogenesis, induced by urethane in mice. The claim that the antagonism between LSD-25 and urethane is “complete” is therefore not substantiated. The results indicate, on the contrary, that the mechanism involved in urethane carcinogenesis is different from that involved in its neurological effects.     

Antagonism of discriminative stimulus effects of Δ9-THC and (R)-methanandamide in rats

Rationale In previous drug discrimination studies we observed surmountable antagonism by Δ⁹-tetrahydrocannabinol (THC) in the presence of constant doses of SR-141716 [N-(piperidin-1-yl)-5-(4-chloro-phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (0.3 and 1 mg/kg), but there was only marginal evidence for surmountable antagonism with combinations of SR-141716 and (R)-methanandamide, a chiral analog of the endocannabioid anandamide. Objective Here we examine antagonism where the cannabinoid CB1 receptor agonist [Δ⁹-THC and (R)-methanandamide] dose is held constant (i.e., the training dose) and the antagonist {i.e., SR-141716 and AM-251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; 2 ml/kg]} dose varied. We also tested the cannabinoid CB2 receptor antagonist SR-144528 {N-[(1S)-endo-1,3,3-trimethylbicyclo(2.2.1)heptan-2-yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide}. Methods Different groups of rats were trained to discriminate between vehicle and three different doses of Δ⁹-THC (1.8, 3, and 5.6 mg/kg, presumably reflecting different efficacy demands) as well as 10 mg/kg (R)-methanandamide. Dose-generalization tests involved different doses of the cannabinoid CB1 receptor agonists. Antagonist tests varied the dose of the antagonist (range: 0.1 and 3 mg/kg for SR-141716 and AM-251, and 1 to 10 mg/kg for SR-144528). Results SR-141716 and AM-251 doses dependently blocked the agonist-induced discriminative stimulus effects. SR-141716 tended to be slightly more potent than AM-251. The effective dose 50 (ED₅₀) of SR-141716 was higher in the 5.6 mg/kg Δ⁹-THC-trained group relative to the two other Δ⁹-THC-trained groups. The cannabinoid CB2 receptor antagonist SR-144528 combined with the training dose of 1.8 mg/kg Δ⁹-THC, as well as when combined with the training dose of 10 mg/kg (R)-methanandamide, did not markedly change drug-appropriate (agonist) responses. Conclusion Data support that the discriminative stimulus effects of (R)-methanandamide and its overlap with the Δ⁹-THC cue are, indeed, CB1 receptor mediated events as revealed in antagonism tests with the selective central CB1 receptor antagonists SR-141716 and AM-251. The activation of cannabinoid CB2 receptors appears to be insignificant for these discriminations.     

Neuroprotective effects of poly(ADP-ribose) polymerase inhibitors in transient focal cerebral ischemia of rats

目的：研究多聚ＡＤＰ核糖多聚酶（ＰＡＲＰ）在局灶性脑缺血再灌注损伤中的作用．方法：雄性Ｗｉｓｔａｒ大鼠用插丝法阻塞大脑中动脉３５ｈ后再灌注，梗塞灶用ＴＴＣ染色显示，图象分析测量；神经功能缺损采用０－５级评分．结果：低剂量ＰＡＲＰ抑制剂３氨基苯甲酰胺（１０ｍｇ·ｋｇ－１）或尼克酰胺（２０ｍｇ·ｋｇ－１）具有明显的神经保护作用，治疗窗近６ｈ；高剂量反而加重脑损伤，特别是尼克酰胺在再灌注起始给药．选择性单ＡＤＰ核糖酰转移酶抑制剂对脑梗塞无明显作用．结论：暂时非完全性抑制ＰＡＲＰ对脑缺血再灌注损伤产生神经保护作用，然而完全抑制该酶（尤其是在再灌注期）则产生损害作用．     

Quantification of haemoglobin binding of 4,4′-methylenebis(2-chloroaniline)(MOCA) in rats

4,4-Methylenebis(2-chloroaniline) (MOCA) is used as a curing agent in the production of polyurethane. MOCA is carcinogenic in experimental animals. Haemoglobin adducts have been proposed as dosimeters of aromatic amines for biological monitoring. A quantitative method to determine the adduct has now been worked out in female Wistar rats dosed per os with 3.82, 14.2 and 16.2 mol/kg¹⁴C-ring labeled MOCA or 0.25 and 0.50 mmol/kg unlabeled MOCA. MOCA bound in decreasing amounts to DNA, RNA and proteins of the lung, liver and kidney. Fractions of 0.19% and 0.026% of the dose were bound to the blood proteins haemoglobin and albumin, respectively. MOCA released by hydrolysis from haemoglobin was determined by HPLC with electrochemical detection or by GC-MS. Albumin did not form any hydrolysable adducts with MOCA.     

Effects of olanzapine infusions to the ventral tegmental area on lordosis and midbrain 3α,5α-THP concentrations in rats

Rationale The progesterone metabolite and neurosteroid 5-pregnane-3-ol-20-one (3,5-THP) facilitates sexual behavior of estradiol-primed rodents through its actions in the ventral tegmental area (VTA). Olanzapine, an atypical antipsychotic, may exert some of its actions by increasing 3,5-THP levels.Objective If olanzapine has effects by increasing 3,5-THP levels, then olanzapine administration to the VTA should facilitate feminine sexual behavior of estradiol-primed rodents concomitant with increasing midbrain levels of 3,5-THP.Methods In experiment 1, ovariectomized rats with bilateral cannulae to the VTA were primed with estradiol at 0 h, infused with olanzapine (10 or 20 g) or vehicle at 47 h, and tested for sexual behavior at 47.5 h. In experiment 2, estradiol-primed ovariectomized rats were infused with olanzapine (10 g) or vehicle, tested for sexual behavior, then tissues were collected for measurement of midbrain progesterone and 3,5-THP, and plasma corticosterone, progesterone, and 3,5-THP. In experiment 3, estradiol-primed, ovariectomized rats were administered progesterone (500 g, SC), tested for sexual behavior, then tissues were collected for midbrain and plasma progesterone and 3,5-THP levels.Results Infusions of 10 or 20 g olanzapine to the VTA significantly increased the incidence and intensity of lordosis, and the occurrence of proceptive and aggressive behaviors. Rats infused with olanzapine to the VTA had significantly greater levels of midbrain 3,5-THP than did vehicle-administered rats. Olanzapine did not increase progesterone or corticosterone levels.Conclusions Olanzapine increases lordosis and midbrain 3,5-THP when infused to the VTA which suggest that olanzapine's behavioral effects may result, in part, through actions of 3,5-THP, independent of progesterone or corticosterone.     

Effects of peptidase inhibitors on anti-nociceptive action of dynorphin-(1–8) in rats

Previous in vitro studies showed that the degradation of dynorphin-(1-8) [dyn-(1-8)] by cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors (PIs), amastatin, captopril and phosphoramidon. In the present investigations, effects of the three PIs on the anti-nociception induced by the intra-third-ventricular (i.t.v.) administration of dyn-(1-8) were examined. The inhibitory effect of dyn-(1-8) on the tail-flick response was increased more than 100-fold by the i.t.v. pretreatment of rats with the three PIs. The inhibition produced by dyn-(1-8) in rats pretreated with any combination of two PIs was significantly smaller than that in rats pretreated with three PIs, indicating that any residual single peptidase could inactivate significant amounts of dyn-(1-8). The antagonistic effectiveness of naloxone, a relatively selective mu-opioid antagonist, indicates that dyn-(1-8)-induced inhibition of tail-flick response in rats pretreated with three PIs is mediated by mu-opioid receptors. Furthermore, mu-receptor-mediated inhibition induced by dyn-(1-8) was significantly greater than that produced by [Met5]-enkephalin in rats pretreated with three PIs. The data obtained in the present investigations together with those obtained in previous studies strongly indicate that dyn-(1-8) not only has well-known kappa-agonist activity but also has high mu-agonist activity.     

Evaluation of GABAergic neuroactive steroid 3α-hydroxy-5α-pregnane-20-one as a neurobiological substrate for the anti-anxiety effect of ethanol in rats

Rationale Acute systemic ethanol administration is known to elevate plasma and cerebral levels of neuroactive steroid 3-hydroxy-5-pregnane-20-one (3, 5-THP; allopregnanolone) to a concentration sufficient to potentiate GABA_A receptors. We have earlier demonstrated that 3, 5-THP mediates the antidepressant-like effect of ethanol in Porsolt forced swim test.Objective The aim of the present study is to explain the relationship between endogenous GABAergic neurosteroids and anxiolytic effect of ethanol in Sprague–Dawley rats.Method The mediation of 3, 5-THP in the anti-anxiety effect of ethanol was assessed by pharmacological interactions of ethanol with various endogenous neurosteroidal modulators and using simulated physiological conditions of altered neurosteroid content in elevated plus maze (EPM) test.Results Pretreatment of 3, 5-THP (0.5–2.5 g/rat, i.c.v.) or neurosteroidogenic agents such as 3, 5-THP precursor progesterone (5 or 10 mg/kg, i.p.), 11- hydroxylase inhibitor metyrapone (50 or 100 mg/kg, i.p.) or the GABA_A receptor agonist muscimol (25 ng/rat, i.c.v.) significantly potentiated the anti-anxiety effect of ethanol (1 g/kg, i.p.). On the other hand, the GABAergic antagonistic neurosteroid dehydroepiandrosterone sulphate (DHEAS) (1 mg/kg, i.p.), the GABA_A receptor blocker bicuculline (1 mg/kg, i.p.), the 5-reductase inhibitor finasteride (50×2 mg/kg, s.c.) or the mitochondrial diazepam binding inhibitory receptor antagonist PK11195 (1 mg/kg, i.p.) reduced ethanol-induced preference of time spent and number of entries into open arms. Anti-anxiety effect of ethanol was abolished in adrenalectomized (ADX) rats as compared to sham-operated control. This ADX-induced blockade was restored by prior systemic injection of progesterone, signifying the contribution of peripheral steroidogenesis in ethanol anxiolysis. Socially isolated animals known to exhibit decreased brain 3, 5-THP and GABA_A receptor functions displayed reduced sensitivity to the effects of ethanol and 3, 5-THP in EPM test.Conclusions Our results demonstrated the contributory role of neuroactive steroid 3, 5-THP in the anti-anxiety effect of ethanol. It is speculated that ethanol-induced modulation of endogenous GABAergic neurosteroids, especially 3, 5-THP, might be crucial pertinent to the etiology of trait anxiety (tension reduction) and ethanol abuse.     

Reduction of circulating and selective limbic brain levels of (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP) following forced swim stress in C57BL/6J mice

Rationale

Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, and GABAergic neuroactive steroids contribute to homeostatic regulation of this circuitry. Acute forced swim stress (FSS) increases plasma, cortical, and hypothalamic (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP) levels in rats. However, there have not been systemic investigations of acute stress on changes in plasma and brain levels of 3α,5α-THP in mouse models.

Objectives

The present experiments aimed to assess circulating and local brain levels of 3α,5α-THP following acute FSS in C57BL/6J mice.

Methods

Mice were exposed to FSS (10 min), and 50 min later, blood and brains were collected. Circulating pregnenolone and 3α,5α-THP levels were assessed in serum. Free-floating brain sections (40 μm, four to five sections/region) were immunostained and analyzed in cortical and limbic brain structures.

Results

FSS decreased circulating 3α,5α-THP (?41.6?±?10.4 %) and reduced 3α,5α-THP immunolabeling in the paraventricular nucleus of the hypothalamus (?15.2?±?5.7 %), lateral amygdala (LA, ?31.1?±?13.4 %), and nucleus accumbens (NAcc) shell (?31.9?±?14.6). Within the LA, vesicular glutamate transporter 1 (VGLUT1) and vesicular GABA transporter were localized in 3α,5α-THP-positively stained cells, while in the NAcc shell, only VGLUT1 was localized in 3α,5α-THP-positively stained cells, suggesting that both glutamatergic and GABAergic cells within the LA are 3α,5α-THP-positive, while in the NAcc shell, 3α,5α-THP only localizes to glutamatergic cells.

Conclusions

The decrease in circulating and brain levels of 3α,5α-THP may be due to alterations in the biosynthesis/metabolism or changes in the regulation of the HPA axis following FSS. Changes in GABAergic neuroactive steroids in response to stress likely mediate functional adaptations in neuronal activity. This may provide a potential targeted therapeutic avenue to address maladaptive stress responsivity.