Bioconversion of antifungal viridin to phytotoxin viridiol by environmental non-viridin producing microorganisms.

Biotransformation of viridin, an antifungal produced by biocontrol agent, with non-viridin producing microorganisms is studied. The results show that some environmental non-targeted microorganisms are able to reduce it in the known phytotoxin viridiol, and its 3-epimer. Consequently, this reduction, which happens in some cases by detoxification mechanism, could be disastrous for the plant in a biocontrol of plant disease. However, a process fermentation/biotransformation could be an efficient approach for the preparation of this phytotoxin.

The prodrug DHED selectively delivers 17ß-estradiol to the brain for treating estrogen-responsive disorders.

Many neurological and psychiatric maladies originate from the deprivation of the human brain from estrogens. However, current hormone therapies cannot be used safely to treat these conditions commonly associated with menopause because of detrimental side effects in the periphery. The latter also prevents the use of the hormone for neuroprotection. We show that a small-molecule bioprecursor prodrug, 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED), converts to 17ß-estradiol in the brain after systemic administration but remains inert in the rest of the body. The localized and rapid formation of estrogen from the prodrug was revealed by a series of in vivo bioanalytical assays and through in vivo imaging in rodents. DHED treatment efficiently alleviated symptoms that originated from brain estrogen deficiency in animal models of surgical menopause and provided neuroprotection in a rat stroke model. Concomitantly, we determined that 17ß-estradiol formed in the brain from DHED elicited changes in gene expression and neuronal morphology identical to those obtained after direct 17ß-estradiol treatment. Together, complementary functional and mechanistic data show that our approach is highly relevant therapeutically, because administration of the prodrug selectively produces estrogen in the brain independently from the route of administration and treatment regimen. Therefore, peripheral responses associated with the use of systemic estrogens, such as stimulation of the uterus and estrogen-responsive tumor growth, were absent. Collectively, our brain-selective prodrug approach may safely provide estrogen neuroprotection and medicate neurological and psychiatric symptoms developing from estrogen deficiency, particularly those encountered after surgical menopause, without the adverse side effects of current hormone therapies.

Genotypes of Fraxinus excelsior with different susceptibility to the ash dieback pathogen Hymenoscyphus pseudoalbidus and their response to the phytotoxin viridiol - a metabolomic and microscopic study.

Eight European ash (Fraxinus excelsior) genotypes with different known susceptibility to Hymenoscyphus pseudoalbidus were tested against the phytotoxin viridiol and their response described at the microscopic and metabolomic level. All ash genotypes were sensitive to the toxin and necrosis was detectable after 24h. Among the three viridiol concentrations used in the experiment, the lowest concentration (14.7µM) yielded markedly lower mean damage scores compared to those resulting from seedlings tested at higher dosages. The highest damage scores were associated with the susceptible ash clones S-101, S-106 and S-125, but also with resistant clone R-104. Three resistant clones (R-131, R-121, and R-118) had lower mean damage scores compared to susceptible clones. Wilting of leaves was more common 48h after treatment and more pronounced on seedlings with high damage scores. The resulting lesions generally lacked browning of tissue and displayed only surface disruption of cells in direct contact with the toxin. A delay in symptom development was evident on all five resistant clones tested with the two higher concentrations of viridiol. LC-HRMS and MS/MS analyses of ash seedling extracts suggest several secoiridoid compounds as well as compounds related to abscisic acid (ABA) to be produced in response to viridiol. ABA-cysteine and xanthoxin were found at significantly higher concentrations in susceptible clones compared to resistant clones after treatment with viridiol, suggesting a primary role of ABA in response to stress. The results observed in this study suggest that genetic resistance to H. pseudoalbidus among ash genotypes may be explained, in part, by the varied response to phytotoxins produced by the fungus.

The neuro-steroid, 5-androstene 3ß,17α diol; induces endoplasmic reticulum stress and autophagy through PERK/eIF2α signaling in malignant glioma cells and transformed fibroblasts.

In this study, we identified a mechanism by which the neuro-steroid, 5-androstene 3ß,17α diol (17α-AED) induces autophagy in human malignant glioma cells and transformed fibroblasts. 17α-AED treatment induced endoplasmic reticulum (ER) stress, identified by the partial activation of an unfolded protein response in T98G, U87MG, U251MG, LN-18, LN-229 and LN-Z308 glioma cell lines. In this regard, there were increased levels of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein of 78kDa transcripts but no splicing of X-box-binding protein 1 mRNA or processing of activating transcription factor-6 in glioma cells treated with the neuro-steroid. 17α-AED induced eukaryotic translational initiation factor 2α (eIF2α) phosphorylation in glioma cells which correlated with microtubule-associated protein-light chain 3 (LC3) conversion from LC3-I to -II. In transformed murine embryonic fibroblasts (MEFs) that are deficient of eIF2α function or T98G glioma cells transfected with a dominant-negative eIF2α construct, 17α-AED induced LC3 conversion was significantly reduced as compared to control cells. Neuro-steroid treatment caused the activation of the eIF2α kinase, protein kinase-like ER kinase (PERK) but not other eIF2α kinases in glioma cells. Moreover, eIF2α phosphorylation and LC3 conversion, in response to 17α-AED treatment, was blocked in MEFs that lacked PERK activity. T98G cells transfected with a dominant-negative PERK construct exhibited an attenuated response to neuro-steroid treatment in terms of decreases in: eIF2α activation; CHOP expression; the incidence of autophagy; and cytotoxicity. These results demonstrate that ER stress is linked to 17α-AED induced autophagy by PERK/eIF2α signaling in human malignant glioma cells and transformed fibroblasts.

Effects of alpha/beta-androstenediol immune regulating hormones on bone remodeling and apoptosis in osteoblasts.

A large body of evidence suggests that the immune system directly impacts bone physiology. We tested whether immune regulating hormones (IRH), 17beta-androstenediol (beta-AED), 7beta,17beta-androstenetriol (beta-AET) or the 17alpha-androstenediol (alpha-AED), and 7alpha,17beta-androstenetriol (alpha-AET) metabolites could directly influence bone remodeling in vitro using human fetal osteoblasts (FOB-9). The impact on bone remodeling was examined by comparing the ratio of RANKL/OPG gene expression in response to AED and AET compounds. The alpha-AED was found to significantly increase in the ratio of RANKL/OPG gene expression and altering the morphology of RANKL stained FOB-9 cells. Cell viability was assessed using a Live/Dead assay. Again alpha-AED was unique in its ability to reduce the proportion of viable cells, and to induce mild apoptosis of FOB-9 cells. Treatment of FOB-9 cells with WY14643, an activator of PPAR-alpha and -gamma, also significantly elevated the percentage of dead cells. This increase was abolished by co-treatment with GW9962, a specific inhibitor of PPAR-gamma. Analysis of PPAR-gamma mRNA by Quantitative RT-PCR and its activation by DNA binding demonstrated that alpha-AED increased PPAR-gamma activation by 19%, while beta-AED conferred a 37% decrease in PPAR-gamma activation. In conclusion, alpha-AED opposed beta-AED by elevating a bone resorption scenario in osteoblast cells. The increase in RANKL/OPG is modulated by an activation of PPAR-gamma that in turn caused mild apoptosis of FOB-9 cells.

Tissue selectivity of the anabolic steroid, 19-nor-4-androstenediol-3beta,17beta-diol in male Sprague Dawley rats: selective stimulation of muscle mass and bone mineral density relative to prostate mass.

Stimulation of prostate growth is a major concern with testosterone therapy in older hypogonadal men. As a result, nonsteroidal selective androgen receptor modulators with anabolic activity but less prostate stimulation are being developed. Anabolic steroids might exhibit similar tissue selectivity. We hypothesized the anabolic steroid 19-nor-4-androstenediol-3beta,17beta-diol (3beta,19-NA) would increase muscle, lean body mass (LBM), and bone mineral density (BMD) with little stimulation of prostate growth. Male Sprague Dawley rats were implanted with SILASTIC brand (Dow Corning, Midland, MI) capsules containing 3beta,19-NA (4, 8, or 16 cm), dihydrotestosterone (DHT) (8 cm), 19-nortestosterone (16 cm), or four empty capsules after undergoing either a sham operation (intact) or orchidectomy (ORX). Serum gonadotropins, measured after 4, 8, or 24 wk of treatment, were significantly lower in 3beta,19-NA-treated vs. untreated, intact, and ORX rats (P < 0.05), and testosterone was lowered by 3beta,19-NA-treatment of intact animals. LBM and BMD were assessed after 20 wk, and 4 wk later, rats were killed for levator ani muscle and prostate weights. Compared with ORX rats, 3beta,19-NA-treated rats had dose-dependent higher levator ani muscle weights, LBM, and BMD, which were similar to intact and DHT-treated rats at the highest 3beta,19-NA dose. In contrast, prostate weights in all 3beta,19-NA-treated groups were similar to ORX rats and lower than intact and DHT- and 19-nortestosterone-treated rats even at the highest 3beta,19-NA dose. In summary, 3beta,19-NA increases muscle and bone mass without significant stimulation of prostate growth, suggesting it may have some properties of a steroidal selective androgen receptor modulator. Anabolic steroids such as 3beta,19-NA should be studied further to determine their mechanisms of tissue selectivity and effects in men.

Bridged androstenediol analogs as ER-beta selective SERMs.

A series of bridged androstenediol derivatives was prepared. The bridged compounds exhibited reduced ER-beta selectivity relative to uncyclized analogs.

Testosterone prohormone supplements.

Testosterone prohormones such as androstenedione, androstenediol, and dehydroepiandrosterone (DHEA) have been heavily marketed as testosterone-enhancing and muscle-building nutritional supplements for the past decade. Concerns over the safety of prohormone supplement use prompted the United States Food and Drug Administration to call for a ban on androstenedione sales, and Congress passed the Anabolic Steroid Control Act of 2004, which classifies androstenedione and 17 other steroids as controlled substances. As of January 2005, these substances cannot be sold without prescription. Here, we summarize the current scientific knowledge regarding the efficacy and safety of prohormone supplementation in humans. We focus primarily on androstenedione, but we also discuss DHEA, androstenediol, 19-nor androstenedione, and 19-nor androstenediol supplements. Contrary to marketing claims, research to date indicates that the use of prohormone nutritional supplements (DHEA, androstenedione, androstenediol, and other steroid hormone supplements) does not produce either anabolic or ergogenic effects in men. Moreover, the use of prohormone nutritional supplements may raise the risk for negative health consequences.

9-epi-Viridiol, a novel cytotoxic furanosteroid from soil fungus Trichoderma virens.

A novel furanosteroid named 9-epi-viridiol (1), along with viridiol (2) and mevalonic acid (3), was isolated from Trichoderma virens. The structure of 1 was verified by combined spectroscopic data (COSY, HSQC, HMBC and NOESY) to be a C-9 epimer of viridiol. 9-epi-Viridiol exhibited cytotoxicity towards HeLa and KB cells with IC50 values of 19 and 50 microg mL(-1), respectively.

Oral andro-related prohormone supplementation: do the potential risks outweigh the benefits?

Androstenedione, 4-androstenediol, 5-androstenediol, 19-norandrostenediol and 19-norandrostenedione are commonly referred to as "Andro" prohormones. Over the last few years, supplementation using these prohormones has been aggressively marketed to the general public. Supplement manufacturers often claim that Andro use improves serum testosterone concentrations, increases muscular strength and muscle mass, helps to reduce body fatness, enhances mood, and improves sexual performance. However, to date, most studies contradict these claims. In contrast, several studies using oral Andro related prohormones show that Andro use can abnormally elevate estrogen related hormones as well as alterations in hormonal markers (i.e., abnormal elevations in serum estrogen) thought to increase a person's risk for developing prostate or pancreatic cancers. In addition, most studies also indicate that significant declines in high-density lipoproteins occur leading to an increased cardiovascular disease risk. Thus, to date, the current research base suggests that Andro prohormone use does not support manufacturer claims. But it does suggest there should be strong concerns regarding long-term oral Andro prohormone use, especially regarding its effects on blood lipids and estrogen hormone profiles.

Effects of norandrostenedione and norandrostenediol in resistance-trained men.

The purpose of this study was to determine the effects of norsteroid supplementation (224 mg of 19-nor-4-androstene-3,17-dione and 120 mg of 19-nor-4-androstene-3,17-diol, total daily dose = 344 mg) on body composition and strength in resistance-trained men. In a placebo-controlled, double-blind, randomized fashion, 10 subjects received the norsteroid (11 capsules containing a combination of both norsteroids) or a placebo for 8 wk (five subjects per group). Each subject participated in resistance training an average of 4 d/wk for the duration of the study. Body composition was determined via dual-energy x-ray absorptiometry. Strength was determined using a one-repetition maximum bench press and a one-repetition maximum biceps curl. With regard to all measures in both groups, there were no significant changes between before and after the study.Therefore, in this small sample of resistance-trained men, 344 mg/d of norsteroid supplementation had no effect on strength or body composition.

Role of hydrophilic interaction in binding of hydroxylated 3-deoxy C(19) steroids to the active site of aromatase.

As part of our investigation into the structure-activity relationship of a novel class of aromatase inhibitors, C(19) steroids having no oxygen function at C-3, we tested aromatase inhibition activity of polar diol compounds 4,19-dihydroxyandrost-5-en-17-ones (25 and 27) and 6,19-dihydroxyandrost-4-en-17-ones (36 and 37). 4alpha,19-Diol 25 was synthesized from tert-butyldimethylsilyoxyandrost-4-ene steroid (9) through its OsO(4) oxidation, giving the 4alpha,5alpha-dihydroxy derivative 12, as a key reaction. Acetylation of 5beta,6alpha-dihydroxy-19-acetate 30 and its 5alpha,6beta-analogue 31 followed by dehydration with SOCl(2) and alkaline hydroxysis gave 6alpha,19-diol 36 and its 6beta-isomer 37, respectively. The stereochemistry of a hydroxy group at C-4 of compound 25 and that at C-6 of compounds 36 and 37 were determined on the basis of (1)H NMR spectroscopy in each case. 4beta,19-Diol 27, previously synthesized, was identified as an extremely powerful competitive inhibitor of aromatase (K(i) = 3.4 nM). In contrast, its 4alpha,19-dihydroxy isomer 25 and other series of diol compounds, 6,19-dihydroxy-4-en-17-one steroids, were moderate to poor competitive inhibitors (K(i) = 110-800 nM). Through this series of analyses, it was concluded that hydrophilic interaction of a 4beta,19-diol function with the active site of aromatase plays a critical role in the tight binding of 3-deoxy-5-ene steroids.

The effects of supplementation with 19-nor-4-androstene-3,17-dione and 19-nor-4-androstene-3,17-diol on body composition and athletic performance in previously weight-trained male athletes.

The purpose of this study was to determine the effects of 8 weeks of norsteroid supplementation on body composition and athletic performance in previously weight-trained males. Subjects were weight and percent body fat matched and randomly assigned to receive either 100 mg of 19-nor-4-androstene-3,17-dione (N-dione) and 56 mg of 19-nor-4-androstene-3,17-diol (N-diol; 156 mg total norsteroid per day), or a placebo (a multivitamin). Each subject participated in resistance training 4 days/week for the duration of the study. Body composition was assessed via dual-energy X-ray absorptiometry. Circumference measures were taken of a relaxed and flexed arm (maximum circumference of the arm), waist (level of umbilicus), and thigh (15 cm proximal to the patella). Strength was determined with a one-repetition maximum bench press, while force and power were determined with a dumbbell bench press (60% body weight) on a Stratec Galileo force platform. Profile of mood states scores were evaluated for vigor and fatigue. There were no significant changes in any of the parameters measured. In conclusion, low-dose supplementation with N-dione and N-diol does not appear to alter body composition, exercise performance, or mood states.

Over-the-counter anabolic steroids 4-androsten-3,17-dione; 4-androsten-3beta,17beta-diol; and 19-nor-4-androsten-3,17-dione: excretion studies in men.

Since the appearance of 4-androsten-3,17-dione (I) as a nutritional supplement in early 1997, we have frequently observed a characteristic deterioration of endogenous steroid profiles in athletes' urine in routine anabolic steroid testing in which concentrations of major endogenous urinary steroids and testosterone exceed normal. Human excretion studies are performed with I and newer, over-the-counter "supplements" 4-androsten-3beta,17beta-diol (II) and 19-nor-4-androsten-3,17-dione (III). Endogenous urinary steroids affected by I and II are androsterone, etiocholanolone, their hydroxylated derivatives 5alpha- and 5beta-androstan-3alpha,17beta-diols, testosterone, and epitestosterone. Their concentrations briefly increase by one to two orders of magnitude and return to normal 24 h after oral administration of I and II. The average male may test positive for testosterone because testosterone concentration rises faster than that of epitestosterone, causing the testosterone/epitestosterone (T/E) ratio to rise above the positive cutoff of 6:1. A remarkable distinction in excretion patterns was observed in eastern Asian men, for whom I and II did not affect urinary concentrations of testosterone and did not increase the T/E ratio. First-pass metabolism deactivates most of the orally administered drugs I and II, rapidly converting them into inactive androsterone and etiocholanolone. Drug II is a more effective testosterone booster because of its different metabolic pathway. After the use of III, a precursor of the potent anabolic nandrolone, high concentrations of norandrosterone and noretiocholanolone appear in urine, similar to nandrolone. These are detectable in urine for 7-10 days after a single oral dose of III (50 mg).

The steroid hormone dehydroepiandrosterone (DHEA) breaks intranasally induced tolerance, when administered at time of systemic immunization.

The induction of intranasal tolerance might be dependent on specific characteristics of mucosal, nose-draining lymph nodes. Such a specific characteristic might lie in the metabolism of the steroid hormone DHEA. Conversion of the prohormone DHEAS into DHEA is dependent on DHEAS-sulphatase activity in lymph nodes. This activity is low in mucosa-draining lymph nodes compared to peripheral lymph nodes, leading to differences in microenvironment. However, administration of DHEA before the induction of intranasal tolerance, could not change tolerance induction. We next determined the effect of DHEA after the induction of intranasally induced tolerance and demonstrated that the steroid hormone and some of its derivatives are able to break tolerance, when administered at time of systemic immunization. These findings might have implications for the regulation of intranasal tolerance and the use of DHEA.

Antiglucocorticoid function of androstenetriol.

The anti-inflammatory and immunosuppressive functions of corticosteroids have been well established and characterized. In contrast, a different group of native steroids, which include dehydroepiandrosterone (DHEA) and two of its metabolites, androstenediol (5-androstene-3 beta-17 beta-diol, AED) and androstenetriol (5-androstene-3 beta-7 beta-17 beta-triol, beta AET), function in vivo to up-regulate host immune response against infections and counteract stress-induced immunosuppression. Indeed, DHEA and particularly, AED and beta AET, have been shown to protect mice from viral, bacterial, and parasitic infections. In vivo, these three hormones are in opposition to the widely demonstrated immunosuppressive action of glucocorticoids, suggesting a possible new immune regulation mechanism. The individual activity in vitro of each of these steroids, i.e. DHEA, AED, and beta AET, on a mitogen-induced mixed splenocyte proliferation assay were determined. The results showed that DHEA suppressed the proliferation of cultures activated with concanavalin A (ConA) or lipopolysaccharide (LPS) in a dose-dependent manner. AED had little influence on the activation response. However, beta AET potentiated the response to both mitogens significantly above control. The regulation of the cytokine secretion, of both interleukin-2 (IL-2) and interleukin-3 (IL-3), from ConA-activated lymphocytes was affected in the same manner. These functions were depressed by DHEA, unaffected by AED, and potently increased by beta AET. Moreover, the classic immunosuppressive effects of hydrocortisone on ConA-induced lymphocyte proliferation, as well as on IL-2 and IL-3 production, were unaffected by being co-cultured with DHEA and only minimally counteracted by AED at high doses. In contrast, co-culturing with beta AET significantly counteracted the immunosuppressive effects of hydrocortisone on lymphocyte proliferation and cytokine production. These data show that in-vivo, DHEA, AED, and beta AET may have some similar functions, while in vitro, their effects are dramatically different from one another. Only beta AET could markedly potentiate the cellular response by increasing lymphocyte activation and counteracting the immnosuppressive activity of hydrocortisone on lymphocyte proliferation and cytokine production.

The effects of dehydroepiandrosterone (DHEA) and its metabolites on the polycystic ovarian condition (PCO): cystogenic changes of rat granulosa cells in vitro.

During mammalian folliculogenesis, granulosa cells (GCs) are initially steroidogenically quiescent, later proliferate, and subsequently commence to hormonally differentiate, first producing estrogen and later, in the preovulatory stage, secreting both estrogen and progesterone. In this study and elsewhere, we have used follicle-stimulating hormone with a combination of growth factors in vitro to simulate the above in vivo conditions. In a previous study, we used dehydroepiandrosterone (DHEA) to accomplish the polycystic ovary condition (PCO) in rats. In the latter model, there were high circulating levels of DHEA and its metabolite, androstenedione. In the present study, we investigated the effects of high levels of DHEA (10(-5) M) and its metabolites, androstenedione, androstenediol and dehydroepiandrosterone sulfate on the quiescent, proliferative, and steroidogenically differentiating stages of GCs cultured in a serum-free medium for up to 10 days. In addition to possessing the regularly occurring organelles, when cultured with the aforementioned androgens, the GCs acquired endoplasmic reticulum of the smooth variety which is associated with steroidogenesis. The radioimmunoassay data showed that GCs cultured in the quiescent and proliferative stages in the presence of the androgens, no longer remain in these stages but proceed to differentiate in a preovulatory direction by producing both estrogen and progesterone. This study supports our hypothesis that high circulating levels of DHEA and/or its metabolites have most effect during the quiescent and proliferative stages of granulosa cells, with regard to their structure and their steroidogenic activities.

In vitro potentiation of lymphocyte activation by dehydroepiandrosterone, androstenediol, and androstenetriol.

Dehydroepiandrosterone (5-androstene-3 beta-ol-17-one, DHEA) has been shown to protect mice from lethal viral and bacterial infections. However, recently, androstenediol (5-androstene-3 beta-17 beta-diol, AED) and androstenetriol (5-androstene-3 beta-7 beta-triol, AET), metabolites of DHEA, have each shown to be more potent endocrine regulators of the immune response. In contrast to glucocorticosteroids, in vivo, these steroid hormones up-regulated the cellular immune response of the host to limit virus-mediated pathology. These experiments first examined the in vitro influences of DHEA, and AED, or AET on mitogen-stimulated activation of murine lymphocytes. From physiologic to pharmacologic doses, DHEA suppressed proliferation of mixed splenocyte cultures activated with Con A or LPS by 20 to 70% whereas AED had little influence on the response. In sharp contrast, AET potentiated the response with both mitogens to 50 to 70% above control. Analogous to these observations was the regulation of IL-2 and IL-3 secretion from Con A-activated lymphocytes by each of these hormones which again was depressed analyzed. The suppressive effects of hydrocortisone on Con A-induced lymphocyte proliferation and cytokine production were strongly counteracted by coculture with AET. DHEA did not counteract hydrocortisone activity whereas AED showed moderate antiglucocorticoid function.