Androstenediol modulates sepsis induced alterations of survival and immune functions in a murine model of sepsis.

BACKGROUND: Recently the benefit of subcutaneously applied dehydroepiandrosterone (DHEA) during sepsis was demonstrated. It was therefore supposed that the impact of DHEA might be induced by its metabolite androstenediol produced via conversion in subcutaneous tissue. Thus we postulate a comparable impact of intravenously applied androstenediol like DHEA. MATERIAL AND METHODS: Male NMRI mice were subjected to sham-operation (laparotomy) or sepsis (cecal ligation and puncture). Animals received saline, DHEA (20 mg/kg/day) subcutaneously, androstenediol (20 mg/kg/day) subcutaneously and androstenediol (10 mg/kg/day) intravenously. During 48 h of sepsis and treatment clinical parameters such as survival and body temperature were observed. Termination of animals was performed 48 hrs after induction of sepsis in order to monitor splenocyte apoptosis (Annexin V binding capacity), cytokine release (IL-10 and TNF-α, ELISA), and immunological capacity by DTH-Reaction (Delayed type of hypersensitivity). RESULTS: Subcutaneous and intravenous androstenediol administration improved the survival rate of septic mice 48 hrs after induction of CLP like subcutaneous administration of DHEA. (86% vs 53%). This effect was paralleled by a restoration of splenocyte proliferation and DTH reaction, a decreased cellular apoptosis rate of splenocytes, and an attenuation of cytokine release. CONCLUSIONS: Administration of androstenediol induces an increased survival rate and improved cellular immune functions in septic mice. This effect was detected independent of the way of administration and is comparable to those effects induced by subcutaneous DHEA administration. With respect to clinical use during critical illness, intravenous administration of androstenediol seems to be an alternative to subcutaneous DHEA administration.

5-AED enhances survival of irradiated mice in a G-CSF-dependent manner, stimulates innate immune cell function, reduces radiation-induced DNA damage and induces genes that modulate cell cycle progression and apoptosis.

The steroid androst-5-ene-3ß,17ß-diol (5-androstenediol, 5-AED) elevates circulating granulocytes and platelets in animals and humans, and enhances survival during the acute radiation syndrome (ARS) in mice and non-human primates. 5-AED promotes survival of irradiated human hematopoietic progenitors in vitro through induction of Nuclear Factor-κB (NFκB)-dependent Granulocyte Colony-Stimulating Factor (G-CSF) expression, and causes elevations of circulating G-CSF and interleukin-6 (IL-6). However, the in vivo cellular and molecular effects of 5-AED are not well understood. The aim of this study was to investigate the mechanisms of action of 5-AED administered subcutaneously (s.c.) to mice 24 h before total body γ- or X-irradiation (TBI). We used neutralizing antibodies, flow cytometric functional assays of circulating innate immune cells, analysis of expression of genes related to cell cycle progression, DNA repair and apoptosis, and assessment of DNA strand breaks with halo-comet assays. Neutralization experiments indicated endogenous G-CSF but not IL-6 was involved in survival enhancement by 5-AED. In keeping with known effects of G-CSF on the innate immune system, s.c. 5-AED stimulated phagocytosis in circulating granulocytes and oxidative burst in monocytes. 5-AED induced expression of both bax and bcl-2 in irradiated animals. Cdkn1a and ddb1, but not gadd45a expression, were upregulated by 5-AED in irradiated mice. S.c. 5-AED administration caused decreased DNA strand breaks in splenocytes from irradiated mice. Our results suggest 5-AED survival enhancement is G-CSF-dependent, and that it stimulates innate immune cell function and reduces radiation-induced DNA damage via induction of genes that modulate cell cycle progression and apoptosis.

Androstenediol exerts salutary effects on chemokine response after trauma-hemorrhage and sepsis in mice.

OBJECTIVES: The pathogenesis of multiple organ dysfunction syndrome and sepsis after polytrauma is related to the posttraumatic immune response and the associated release of inflammatory mediators. There exists a gender dimorphism in the posttraumatic host response. Sex steroids are believed to beneficially modulate the posttraumatic immune response. The specific effect of androstenediol on chemokines after trauma is unknown. We investigated whether the application of androstenediol has an effect on plasma chemokine levels and the associated remote organ damage in a two-hit mouse-model of trauma-hemorrhage, cecal ligation, and cecal puncture. MATERIALS AND METHODS: Traumatic hemorrhage was induced followed by androstenediol application and volume resuscitation. Thereafter, androstenediol was given once daily in combination with a vehicle (Intralipid). The control group was injected with a solution containing only the vehicle at the same time points as the treatment groups' androstenediol applications. Sepsis was induced by cecal ligation and cecal puncture 48 hours afterward. Four hours after cecal ligation and cecal puncture, plasma measurements of chemokines were performed. Pulmonary infiltration by polymorphonuclear lymphocytes was measured by immunhistochemical staining and myeloperoxidase measurements were taken. RESULTS: Application of androstenediol led to significantly decreased monocyte chemoattractant protein-1, monocyte chemoattractant protein-3, macrophage inflammatory protein-1α, and macrophage inflammatory protein-1ß levels compared with the control animals after trauma-hemorrhage, cecal ligation, and cecal puncture (P < 0.05). Pulmonary infiltration and myeloperoxidase activity were significantly decreased in androstenediol-treated animals (P < 0.05). CONCLUSION: Androstenediol modulates the immune response after trauma-hemorrhage, cecal ligation, and cecal puncture by reducing systemic chemokine levels, which are known to direct immune cells into the tissue possibly leading to organ damage. Androstenediol represents a potential therapeutic agent after major trauma in high-risk patients.

The potential of urinary androstdiene markers to identify 4-androstenediol (4-ADIOL) administration in athletes.

Doping control laboratories accredited by the World Anti-Doping Agency (WADA) require criteria that allow endogenous steroids to be distinguished from their synthetic analogues in urine. Methodology based on "looking outside the metabolic box" was used in this study to identify diagnostic urinary markers of 4-androstenediol (4-ADIOL) administration. Androst-2,4-diene-17-one and androst-3,5-diene-17-one are proposed to be formed in urine from acid-catalyzed hydrolysis of 4-ADIOL sulfoconjugate, a major phase II metabolic product of 4-ADIOL. The presence of these markers in the routine gas chromatography-mass spectrometry (GC-MS) steroid screen was suitable to identify samples requiring confirmation by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) - to measure the carbon isotope ratio (δ(13)C) of the androstdiene markers and confirm their likely synthetic origin based on depleted (13)C content.

Preliminary clinical findings on NEUMUNE as a potential treatment for acute radiation syndrome.

5-androstenediol (5-AED) has been advanced as a possible countermeasure for treating the haematological component of acute radiation syndrome (ARS). It has been used in animal models to stimulate both innate and adaptive immunity and treat infection and radiation-induced immune suppression. We here report on the safety, tolerability and haematologic activity of 5-AED in four double-blinded, randomized, placebo-controlled studies on healthy adults including elderly subjects. A 5-AED injectable suspension formulation (NEUMUNE) or placebo was administered intramuscularly as either a single injection, or once daily for five consecutive days at doses of 50, 100, 200 or 400 mg. Subjects (n = 129) were randomized to receive NEUMUNE (n = 95) or the placebo (n = 34). NEUMUNE was generally well-tolerated; the most frequent adverse events were local injection site reactions (n = 104, 81%) that were transient, dose-volume dependent, mild to moderate in severity, and that resolved over the course of the study. Blood chemistries revealed a transient increase (up to 28%) in creatine phosphokinase and C-reactive protein levels consistent with intramuscular injection and injection site irritation. The blood concentration profile of 5-AED is consistent with a depot formulation that increases in disproportionate increments following each dose. NEUMUNE significantly increased circulating neutrophils (p < 0.001) and platelets (p < 0.001) in the peripheral blood of adult and elderly subjects. A dose-response relationship was identified. Findings suggest that parenteral administration of 5-AED in aqueous suspension may be a safe and effective means to stimulate innate immunity and alleviate neutropenia and thrombocytopenia associated with ARS.

Androstenediol administration after trauma-hemorrhage attenuates inflammatory response, reduces organ damage, and improves survival following sepsis.

Although androstenediol (adiol or 5-androstene-3beta,17beta-diol), a metabolite of dehydroepiandrosterone (DHEA), has protective effects following trauma-hemorrhage (T-H), it remains unknown whether administration of adiol has any salutary effects on the inflammatory response and outcome following a combined insult of T-H and sepsis. Male rats underwent T-H shock [mean arterial pressure (MAP) 40 mmHg for 90 min] followed by resuscitation. Adiol (1 mg/kg body wt) or vehicle was administered at the end of resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) at 20 h after T-H or sham operation. Five hours after CLP, plasma and tissue samples were analyzed for cytokines (IL-6 and IL-10), MPO, neutrophil chemotactic factor (CINC-3), and liver injury (alanine aminotransferase and lactate dehydrogenase). In another group of rats, the gangrenous cecum was removed at 10 h after CLP, the cavity was irrigated with warm saline and closed in layers, and mortality was recorded over 10 days. T-H followed by CLP produced a significant elevation in plasma IL-6 and IL-10 levels, enhanced neutrophil cell activation, and resulted in liver injury. Adiol administration prevented the increase in cytokine production, neutrophil cell activation, and attenuated liver injury. Moreover, rats subjected to the combined insult, receiving vehicle or adiol, had a 50% and 6% mortality, respectively. Since adiol administration suppresses proinflammatory cytokines, reduces liver damage, and decreases mortality after the combined insult of T-H and sepsis, this agent appears to be a novel adjunct to fluid resuscitation for decreasing T-H-induced septic complications and mortality.

Acute resistance exercise does not change the hormonal response to sublingual androstenediol intake.

Sublingual intake of 21.4 mg androstenediol increases serum testosterone concentrations whereas swallowing 200 mg androstenediol does not. The duration of increase in serum testosterone following sublingual androstenediol (SL-DIOL) is unknown. Resistance exercise (EX) following SL-DIOL may cause larger increases in serum estradiol concentrations than while at rest. This project evaluated the duration of change in, and the effects of acute EX on, the hormonal response to SL-DIOL. Six young resistance trained males consumed either placebo (PL) or SL-DIOL before a single session of EX or no exercise (Rest) in a random, double blind, crossover manner (for a total of four trials). Blood samples were collected before supplementation, and at 60, 120, 180, 240, 480, and 720 min post-supplementation, with the exercise occurring between 60 and 120 min. The serum [total testosterone] increased (P < 0.05) at 60 min similarly in SL-DIOL-EX and SL-DIOL-Rest by approximately 115%, and at 120 min by approximately 107% with no differences due to exercise. The serum [estradiol] increased (P < 0.05) similarly in SL-DIOL-EX and SL-DIOL-Rest by approximately 33% at 60 min and approximately 45% at 120 min, with no differences due to exercise. Serum [testosterone] returned to baseline by 240 min and serum [estradiol] returned to baseline by 720 min post-intake. These findings indicate that SL-DIOL acutely elevates serum testosterone and estradiol concentrations, that EX does not alter the endocrine response to SL-DIOL, and that the increases in serum estradiol last between 480 and 720 min while the increases in serum testosterone last <240 min following acute SL-DIOL intake.

Molecular specificity of 5-androstenediol as a systemic radioprotectant in mice.

We compared in vivo radioprotective efficacy of 5-androstenediol (5-AED) to that of ten other steroids: 17alpha-androstenediol, dehydroepiandrosterone, 5-androstenetriol (AET), 4-androstenedione (AND), testosterone, estradiol, fluasterone, 16alpha-bromoepiandrosterone, 16alpha-fluoro-androst-5-en-17alpha-ol (alpha-fluorohydrin, AFH), and 16alpha-fluoro-androst-5-en-17beta-ol (beta-fluorohydrin). Steroids were administered 24 or 48 hr before, or 1 hr after, whole-body gamma-irradiation. Two days after irradiation at 3 Gy, blood elements were counted. In addition, after irradiation at 9-12.5 Gy, survival was recorded for 30 days. The results showed radioprotective efficacy was specific for 5-AED. One other steroid, AFH, demonstrated appreciable survival effects but was less efficacious than 5-AED. AND and AET produced slight enhancement of survival in some experiments. This is the first demonstration that the prophylactic window for survival enhancement by 1 subcutaneous (s.c.) injection of 5-AED is as long as 48 hr in mice. Moreover, the results indicate that 1 s.c. injection of 5-AED 1 hr after irradiation is much less effective than 1 injection 24-48 hr before irradiation. Comparing the molecular features of steroids with radioprotective efficacy leads to the following conclusions: 1) these effects are due to interaction with specific receptors, since s.c. injection of extremely similar molecules with the same physicochemical properties as 5-AED were not radioprotective; 2) the 17-hydroxyl group is essential; 3) this group must be in the beta configuration in the absence of nearby side groups; 4) a halogen atom at 16 changes the 17-hydroxyl specificity to alpha; 5) the 3beta-hydroxyl group is not essential; 6) addition of a 7beta-hydroxyl group is deleterious; and 7) the effects are not due to activation of sex steroid receptors.

Effects of whole-body gamma irradiation and 5-androstenediol administration on serum G-CSF.

5-Androstenediol (5-AED) is a natural circulating adrenocortical steroid hormone that interconverts in vivo with other members of the 5-androstene family of steroids: dehydroepiandrosterone and 5-androstenetriol. These steroids stimulate immune responses and resistance to infection. 5-AED has been identified as a systemic radiation countermeasure that enhances survival in mice exposed to gamma irradiation and ameliorates radiation-induced neutropenia in mice and nonhuman primates. 5-AED mitigates radiation-induced decreases in platelets, natural killer (NK) cells, red blood cells, and monocytes. Administration of 5-AED causes functional activation of circulating granulocytes (phagocytic ability), monocytes (oxidative burst), and NK cells (surface CD11b expression). The effects of 5-AED on survival and hematological parameters are consistent with induction of hematopoietic cytokines. To test this hypothesis, we measured serum cytokines by ELISA, Luminex, and a cytokine array. A cytokine array was used for 62 different cytokines, chemokines, growth factors, and soluble receptors. 5-AED caused significant increases in circulating granulocyte colony-stimulating factor (G-CSF) in irradiated and unirradiated animals as observed with ELISA and Luminex. The cytokine array results suggest induction of G-CSF and additional cytokines, and related molecules. Since G-CSF is an important hematopoietic cytokine, the results support our hypothesis that the previously observed increases in numbers of hematopoietic progenitors, circulating innate immune cells and platelets, and functional activation of granulocytes, monocytes, and NK cells result from a cytokine cascade induced by 5-AED.

Virilization of young children after topical androgen use by their parents.

Children were virilized by contact with adults using cutaneous steroid preparations. Parents were unaware of the dangers of passive transfer. Laboratory data were consistent with exogenous androgen exposure. Each child had opportunity for passive exposure, and discontinuation of contact resulted in a decrease of androgen levels or regression of symptoms.

Acute hormonal response to sublingual androstenediol intake in young men.

The effectiveness of orally ingested androstenediol in raising serum testosterone concentrations may be limited because of hepatic breakdown of the ingested androgens. Because androstenediol administered sublingually with cyclodextrin bypasses first-pass hepatic catabolism, we evaluated the acute hormonal response to sublingual cyclodextrin androstenediol supplement in young men. Eight men (22.9 +/- 1.2 yr) experienced in strength training consumed either 20 mg androstenediol in a sublingual cyclodextrin tablet (Sl Diol) or placebo (Pl) separated by at least 1 wk in a randomized, double-blind, crossover manner. Blood samples were collected before supplementation and at 30-min intervals for 3 h after supplementation. Serum hormone concentrations did not change with Pl. Serum androstenedione concentrations were increased (P < 0.05) above baseline (11.2 +/- 1.1 nmol/l) with Sl Diol from 60 to 180 min after intake and reached a peak concentration of 25.2 +/- 2.9 nmol/l at 120 min. Serum free testosterone concentrations were increased from 86.2 +/- 9.1 pmol/l with Sl Diol from 30 to 180 min and reached a peak concentration of 175.4 +/- 12.2 pmol/l at 60 min. Serum total testosterone concentrations increased above basal (25.6 +/- 2.3 nmol/l) from 30 to 180 min with Sl Diol and reached a peak concentration of 47.9 + 2.9 nmol/l at 60 min. Serum estradiol concentrations were elevated (P < 0.05) above baseline (0.08 +/- 0.01 nmol/l) from 30 to 180 min with Sl Diol and reached 0.14 +/- 0.02 nmol/l at 180 min. These data indicate that sublingual cyclodextrin androstenediol intake increases serum androstenedione, free testosterone, total testosterone, and estradiol concentrations.

Phenotypic adaptations in human muscle fibers 6 and 24 wk after spinal cord injury.

The effects of spinal cord injury (SCI) on the profile of sarco(endo) plasmic reticulum calcium-ATPase (SERCA) and myosin heavy chain (MHC) isoforms in individual vastus lateralis (VL) muscle fibers were determined. Biopsies from the VL were obtained from SCI subjects 6 and 24 wk postinjury (n = 6). Biopsies from nondisabled (ND) subjects were obtained at two time points 18 wk apart (n = 4). In ND subjects, the proportions of VL fibers containing MHC I, MHC IIa, and MHC IIx were 46 +/- 3, 53 +/- 3, and 1 +/- 1%, respectively. Most MHC I fibers contained SERCA2. Most MHC IIa fibers contained SERCA1. All MHC IIx fibers contained SERCA1 exclusively. SCI resulted in significant increases in fibers with MHC IIx (14 +/- 4% at 6 wk and 16 +/- 2% at 24 wk). In addition, SCI resulted in high proportions of MHC I and MHC IIa fibers with both SERCA isoforms (29% at 6 wk and 54% at 24 wk for MHC I fibers and 16% at 6 wk and 38% at 24 wk for MHC IIa fibers). Thus high proportions of VL fibers were mismatched for SERCA and MHC isoforms after SCI (19 +/- 3% at 6 wk and 36 +/- 9% at 24 wk) compared with only ~5% in ND subjects. These data suggest that, in the early time period following SCI, fast fiber isoforms of both SERCA and MHC are elevated disproportionately, resulting in fibers that are mismatched for SERCA and MHC isoforms. Thus the adaptations in SERCA and MHC isoforms appear to occur independently.

Radioprotective efficacy and acute toxicity of 5-androstenediol after subcutaneous or oral administration in mice.

We previously showed that one subcutaneous (sc) injection of 5-androstene-3beta,17beta-diol (AED) stimulated the innate immune system in mice and prevented mortality due to hemopoietic suppression after whole-body ionizing irradiation with gamma rays. In the present study, we tested whether there was any significant toxicity in mice that might hinder development of this steroid for human use. There were no indications of toxicity in chemical analyses of serum after sc doses as high as 4000 mg/kg. At this dose, 2 of 54 mice died when given AED alone. When 4800 mg/kg was given orally, no deaths resulted. The only adverse findings attributed to AED administration were 1) a moderate elevation of granulocytes in abdominal organs and fat after sc injections of 320 mg/kg; and 2) occasional wasting of skin over the injection site in female B6D2F1 but not male C3H/HeN mice. Significant weight loss (6%) was observed after sc injections of 320 mg/kg but not 160 or 80 mg/kg. When male C3H/HeN mice were injected sc with AED at doses of 0-200 mg/kg 24 h before whole body gamma-irradiation (9 Gy), a significant improvement in survival was observed at doses as low as 5 mg/kg. Oral administration of AED produced significant survival enhancement at a dose of 1600 mg/kg. We conclude that the radioprotective efficacy of AED is accompanied by low toxicity.

Endocrine and lipid responses to chronic androstenediol-herbal supplementation in 30 to 58 year old men.

OBJECTIVE: The effectiveness of an androgenic nutritional supplement designed to enhance serum testosterone concentrations and prevent the formation of dihydrotestosterone and estrogen was investigated in healthy 3 to 58 year old men. DESIGN: Subjects were randomly assigned to consume a nutritional supplement (AND-HB) containing 300-mg androstenediol, 480-mg saw palmetto, 450-mg indole-3-carbinol, 300-mg chrysin, 1,500 mg gamma-linolenic acid and 1.350-mg Tribulus terrestris per day (n = 28), or placebo (n = 27) for 28 days. Subjects were stratified into age groups to represent the fourth (30 year olds, n = 20), fifth (40 year olds, n = 20) and sixth (50 year olds, n = 16) decades of life. MEASUREMENTS: Serum free testosterone, total testosterone, androstenedione, dihydrotestosterone, estradiol, prostate specific antigen and lipid concentrations were measured before supplementation and weekly for four weeks. RESULTS: Basal serum total testosterone, estradiol, and prostate specific antigen (PSA) concentrations were not different between age groups. Basal serum free testosterone concentrations were higher (p < 0.05) in the 30- (70.5 +/- 3.6 pmol/L) than in the 50 year olds (50.8 +/- 4.5 pmol/L). Basal serum androstenedione and dihydrotestosterone (DHT) concentrations were significantly higher in the 30- (for androstenedione and DHT, respectively, 10.4 +/- 0.6 nmol/L and 2198.2 +/- 166.5 pmol/L) than in the 40- (6.8 +/- 0.5 nmol/L and 1736.8 +/- 152.0 pmol/L) or 50 year olds (6.0 +/- 0.7 nmol/L and 1983.7 +/- 147.8 pmol/L). Basal serum hormone concentrations did not differ between the treatment groups. Serum concentrations of total testosterone and PSA were unchanged by supplementation. Ingestion of AND-HB resulted in increased (p < 0.05) serum androstenedione (174%), free testosterone (37%), DHT (57%) and estradiol (86%) throughout the four weeks. There was no relationship between the increases in serum free testosterone, androstenedione, DHT, or estradiol and age (r2 = 0.08, 0.03, 0.05 and 0.02, respectively). Serum HDL-C concentrations were reduced (p < 0.05) by 0.14 mmol/L in AND-HB. CONCLUSIONS: These data indicate that ingestion of androstenediol combined with herbal products does not prevent the formation of estradiol and dihydrotestosterone.

Endocrine regulation of murine macrophage function: effects of dehydroepiandrosterone, androstenediol, and androstenetriol.

In these studies, the in vitro influences of dehydroepiandrosterone (DHEA), androstenediol (AED), and androstenetriol (AET) on proinflammatory cytokine production from macrophages was examined. From physiologic to pharmacologic doses, DHEA suppressed secretion of each pro-inflammatory cytokine while AED had little influence on the responses. In sharp contrast, AET augmented TNF-alpha and IL-1 secretion while not influencing IL-6 production. Furthermore, the antiglucocorticoid activity of DHEA, AED, and AET was also investigated. Co-culture with AET counteracted the down-regulatory effect of hydrocortisone on LPS-induced TNF-alpha and IL-1 secretion. These data imply that AET is capable of regulating cytokine secretion from macrophages and may function to counterbalance glucocorticoid function.

Successful treatment of vitiligo with a sex steroid-thyroid hormone mixture.

Four patients with generalized vitiligo were successfully treated by oral administration of a sex steroid-thyroid hormone (Metharmon-F, 2 tablets daily). Histopathologically, the repigmented skin showed increased numbers of melanocytes and melanin granules in the keratinocytes.