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The steroid androst-5-ene-3beta,17beta-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-kappaB (NFkappaB)-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 gamma 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., Published in Journal of Radiation Research Advance Access 2012, 00, p1-14, published July 22, 2012. The original document contains color images.

Presented at the Radiation Countermeasures Symposium held in Bethesda, Maryland on 15 June 2011.

Background: The animal efficacy rule addressing development of drugs for selected disease categories has pointed out the need to develop alternative large animal models. Based on this rule, the pathophysiology of the disease in the animal model must be well characterized and must reflect that in humans. So far, manifestations of the acute radiation syndrome (ARS) have been extensively studied only in two large animal models, the non-human primate (NHP) and the canine. We are evaluating the suitability of the minipig as an additional large animal model for development of radiation countermeasures. We have previously shown that the Gottingen minipig manifests hematopoietic ARS phases and symptoms similar to those observed in canines, NHPs, and humans. Principal Findings: We establish here the LD50/30 dose (radiation dose at which 50% of the animals succumb within 30 days), and show that at this dose the time of nadir and the duration of cytopenia resemble those observed for NHP and canines, and mimic closely the kinetics of blood cell depletion and recovery in human patients with reversible hematopoietic damage (H3 category, METREPOL approach). No signs of GI damage in terms of diarrhea or shortening of villi were observed at doses up to 1.9 Gy. Platelet counts at days 10 and 14, number of days to reach critical platelet values duration of thrombocytopenia, neutrophil stress response at 3 hours and count at 14 days, and CRP-to-platelet ratio were correlated with survival. The ratios between neutrophils, lymphocytes and platelets were significantly correlated with exposure to irradiation at different time intervals. Significance: As a non-rodent animal model, the minipig offers a useful alternative to NHP and canines, with attractive features including ARS resembling human ARS, cost, and regulatory acceptability. Use of the minipig may allow accelerated development of radiation countermeasures., Published in PLoS ONE, v6 n9 e25210 p1-8, September 2011. Sponsored in part by the National Institute of Allergy and Infectious Diseases (NIAID).

Exposure to "-radiation causes rapid hematopoietic cell apoptosis and bone marrow suppression. However, there are no approved radiation countermeasures for the acute radiation syndrome. In this report, we demonstrate that natural delta-tocotrienol, one of the isomers of vitamin E, significantly enhanced survival in total body lethally irradiated mice. We explored the effects and mechanisms of delta-tocotrienol on hematopoietic progenitor cell survival after " -irradiation in both in vivo and in vitro experiments. Design and Methods. CD2F1 mice and human hematopoietic progenitor CD34+ cells were treated with delta-tocotrienol or vehicle control 24 h before or 6 h after " -irradiation. Effects of delta-tocotrienol on hematopoietic progenitor cell survival and regeneration were evaluated by clonogenicity, flow cytometry, and bone marrow histochemical staining. Delta-tocotrienol and "- irradiation-induced signal regulatory activities were assessed by immunofluorescence staining immunoblotting and siRNA assay. Results. Delta-tocotrienol displayed significant radioprotective effects. A single injection of delta-tocotrienol protected 100% of CD2F1 mice from total body irradiation-induced death as measured by 30-day post-irradiation survival. Delta-tocotrienol increased cell survival, and regeneration of hematopoietic microfoci and lineage-negative/Sca-1-positive/ckit-positive stem and progenitor cells in irradiated mouse bone marrow, and protected human CD34+ cells from radiation?induced damage. Delta-tocotrienol activated Erk1/2 phosphorylation and significantly inhibited formation of DNA-damage marker "-H2AX foci. In addition, delta-tocotrienol upregulated mTOR and phosphorylation of its downstream effector 4EBP-1. These alterations were associated with activation of mRNA translation regulator eIF4E and ribosomal protein S6 which is resp, Pub. in Haematologica, p1-37, 2010. ISSN: 0390-6078, 1592-8721. Sponsored in part by Defense Threat Reducation Agency grant no. G1B2BP and AFFRI grant no. G2B2DB.

5-Androstenediol stimulates hematopoiesis and enhances survival in animals exposed to ionizing radiation (IR), suggesting this steroid may act on hematopoietic progenitor cells. We used gamma-irradiated primary human CD34+ hematopoietic progenitor cells to show 5-AED protects hematopoietic cells from IR damage, as shown by enhanced cell survival clonogenicity, proliferation, and differentiation. Unlike in tumor cells, IR did not induce NF-kappa B(NFkB) activation in primary progenitors. However, IR stimulated IkB(sub Beta) release from NFkB/IkB complexes and caused NFkB1 (p50) degradation. 5-AED stabilized NFkB1 in irradiated cells, as well as inducing NFkB gene expression and NFkB activation (DNA binding). 5-AED stimulated interleukin-6 (IL-6) and granulocyte colony-stimulating factor (GCSF) secretion. The survival-enhancing effects of 5-AED on clonogenic cells were abrogated by siRNA inhibition of NFkB gene expression, and also by neutralization of G-CSF with antibody. The effects of 5-AED on survival and G-CSF secretion were blocked by the NFkB inhibitor MG132. 5-AED had no effect on accumulation of the pro-apoptotic factor p53 after IR, as determined by Western blot. The results indicate that NFkB1 degradation after IR may be responsible for the radiation sensitivity of CD34+ cells, as compared to tumor cells. 5-AED exerts survival-enhancing effects on irradiated human hematopoietic progenitor cells via induction, stabilization, and activation of NFkB, which results in increased secretion of hematopoietic growth factor G-CSF.

Lewis rats display a hyporesponsive hypothalamo-pituitary-adrenocortical (HPA) axis, overproduction of cytokines, and susceptibility to inflammatory disease. The Lewis corticotropin-releasing hormone (CRH) neurosecretory system contains normal numbers of vasopressin (VP) deficient axon varicosities but abnormally sparse vasopressin-containing varicosities in the external zone of the median eminence, compared to the normoresponsive Sprague Dawley (SD), Wistar, and Fischer 344 strains. Since vasopressin may act as a thyrotropin-releasing factor, we hypothesized that thyroid axis responsivity may be altered in Lewis rats. T(sub 3), T(sub 4), and the thyroid-stimulating hormone (TSH) were measured by radioimmunoassay, and free T(sub 4) by equilibrium dialysis in adult male Lewis and SD rats. Exposure to cold (5 deg C) for 1 h induced significant increases in T(sub 3), T(sub 4), and TSH levels in Lewis rats but not in SD rats. Ninety min of insulin-induced hypoglycemia (1 IU/kg, ip) caused a significant T(sub 3) increase in Lewis rats and a significant T(sub 4) increase in SD rats. Two h after intraponeal administration of lipopolysaccharide (LPS; (0.25 or 0.75 mg/kg)), T(sub 4) levels fell significantly in Lewis rats but not in SD rats. TSH decreases were significant in Lewis rats after 0.75 mg/kg LPS and in SD rats after 0.25 mg/kg. Baseline hormone levels were generally higher in Lewis rats; the differences were significant for T(sub 3) and T(sub 4) in the insulin experiments and for T(sub 3), T(sub 4), and free T(sub 4) in the LPS experiments. The data suggest that reduced inhibition from the adrenocortical axis in Lewis rats leads to hyperresponsivity of the thyroid axis to cold and greater LPS-induced decreases in T(sub 4) levels, probably due to an exaggerated inhibitory cytokine response.

Administration of lipopolysaccharide (LPS) results in activation of the hypothalamic-pituitary-adrenal axis. LPS induces the release of a number of proinflammatory cytokines, I.E. interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF), which activate the hypothalamic-pituitary-adrenal axis as well and may mediate the effects of LPS. Variations in the kinetics of appearance of IL- 1, TNF, and IL-6 after LPS challenge suggested that these cytokines may play different roles at different times. To elucidate the mutual dependence and contribution of individual cytokines in the course of LPS-induced ACTH release, we used blocking antibodies to IL-6, TNF, and the IL-1 receptor., Availability: Pub. in Endocrinology, v132 n3 p946-952 1993. Available only to DTIC users. No copies furnished by NTIS.

This report describes for the first time the cellular and subcellular localization of estrogen sulfotransferase (EST) as well as the subcellular localization of hydroxysteroid sulfotransferase (HST) in the mammalian adrenal cortex. A 34-kilodalton EST and two HSTs with 3 alpha- and 3 Beta-hydroxysteroid substrate specificities (32 and 33 kilodaltons, respectively) were previously purified from guinea pig adrenal cortex and characterized. Western blots were used to establish that two antisera generated against EST were highly specific for EST, whereas three antisera generated against the HSTs were highly specific for the HSTs, but did not distinguish between the 3 alpha- and 3 Beta HSTs. Light and electron microscopic immunoperoxidase labeling with these antisera revealed that the sulfortransferases were expressed only within the ACTH- responsive layers of the guinea pig adrenal cortex, with EST localized to zona fasciculata and zona reticularis cells, and the HSTs confined to the zona reticularis. No labeling was detected in the zona glomerulosa or adrenal medulla. EST was concentrated in cell nuclei; sparse labeling was distributed throughout the cytoplasm. HST labeling was intense in smooth endoplasmic reticulum of zona reticularis cells, but was absent from nuclei.