Acute toxicity of subcutaneously administered vitamin E isomers delta- and gamma-tocotrienol in mice.

The toxicity of parenterally administered vitamin E isomers, delta-tocotrienol (DT3) and gamma-tocotrienol (GT3), was evaluated in male and female CD2F1 mice. In an acute toxicity study, a single dose of DT3 or GT3 was administered subcutaneously in a dose range of 200 to 800 mg/kg. A mild to moderately severe dermatitis was observed clinically and microscopically in animals at the injection site at doses above 200 mg/kg. The severity of the reaction was reduced when the drug concentration was lowered. Neither drug produced detectable toxic effects in any other tissue at the doses tested. Based on histopathological analysis for both DT3 and GT3, and macroscopic observations of inflammation at the injection site, a dose of 300 mg/kg was selected as the lowest toxic dose in a 30-day toxicity study performed in male mice. At this dose, a mild skin irritation occurred at the injection site that recovered completely by the end of the experimental period. At a dose of 300 mg/kg of DT3 or GT3, no adverse effects were observed in any tissues or organs.

Mechanisms of Radiation Bystander and Non-Targeted Effects: Implications to Radiation Carcinogenesis and Radiotherapy.

BACKGROUND: Knowledge of radiobiology is of paramount importance to be able to grasp and have an in-depth understanding of the consequences of ionizing radiation. One of the most important effects of this physical stressor's interaction to targeted and non-targeted cells, tissues and organs is on the late effects on the development of primary and secondary cancers. Thus, an in-depth understanding of the mechanisms of radiation carcinogenesis remains to be elucidated, and some studies have demonstrated or proposed a role of non-targeted effect in excess risk of cancer incidence. The non-targeted effect in radiobiology refers to a dynamic complex response in non-irradiated tissues caused by the release of presumably of clastogenic factors from irradiated cells. Although, most of these responses in non-targeted tissues have marked similarities to irradiated tissues, other studies have shown some differences. Also, the non-targeted effect has shown sex and tissue specificity that are seen in irradiated tissues too. So far, several studies have been conducted to depict mechanisms that may be involved in this phenomenon. Epigenetic dysfunctions, DNA damage and cell death are responsible for initiation of several signaling pathways that finally result in secretion of clastogenic factors. Moreover, studies have shown that damage to both nucleus and mitochondrial DNA, membrane and some organelles is involved. Oxidized DNA associated with other cell death factors stimulates secretion of inflammatory as well as some anti-inflammatory cytokines from irradiated area. Additionally, oxidative stress that results in damage to cellular structures to include cell membranes can affect secretion of exosomes and miRNAs. These bystander effect exogenous mediators migrate to distant tissues and stimulate various signaling pathways which can lead to changes in immune responses, epigenetic modulations and radiation carcinogenesis. CONCLUSION: In this review, we focus on descriptive and hierarchical events with emphasis on the molecular and functional interactions of ionizing radiation with cells to the mechanisms involved in cancer induction in non-targeted tissues.

Radiation protection and mitigation potential of phenylbutyrate: delivered via oral administration.

PURPOSE: Phenylbutyrate (PB), a histone deacetylase inhibitor (HDACi) has demonstrated radiation protection in both in vitro and in vivo models. Studies previously demonstrated that PB and other HDAC inhibitors could inhibit radiation lethality in vivo by subcutaneous (s.c) injection. The objective of this study was to test the ability of oral PB treatment to protect against or to mitigate acute gamma radiation-induced lethality in vivo. MATERIALS AND METHODS: Human osteoblasts cells were used to evaluate radiation survival when PB was delivered pre- or post-radiation. A 30-day radiation lethality study was used to assess the radioprotective (pre-radiation) and radiomitigative (post-radiation) capability of PB. Possible mechanisms evaluated were antioxidant activity effects, HDAC inhibition, DNA damage, and hematological recovery. RESULTS: Treatment of HOS cells with PB 50 µM either before or after radiation increased radiation resistance as assessed by clonogenic survival. Western blot studies showed that PB treatment acetylated histones in vivo and ameliorated the radiation-induced reduction in acetylated histone-4 (H4). Pre-radiation oral administration of PB (10 mg/kg) provided radioprotection against gamma radiation (7-11.5 Gy) with a dose reduction factor of 1.25 (p = 0.001). PB oral administration post-radiation provided moderate radiation mitigation against gamma radiation (7-11.5 Gy) and demonstrated a dose reduction factor of 1.18 (p = 0.05). PB pre-radiation and post-radiation treatment was associated with significant elevations in neutrophils and platelets and attenuation of DNA damage. CONCLUSIONS: These results indicate that oral PB has potential as a radiation protector and a radiation mitigator and that potential mechanisms of action include attenuation of DNA damage, antioxidant activity, and bone marrow protection.

Depleted uranium-uranyl chloride induces apoptosis in mouse J774 macrophages.

Depleted uranium entering the body as a result of inhalation or embedded fragments becomes associated to a great extent with macrophages. As part of our continuing studies on the health effects of internalized depleted uranium, we investigated the effect of soluble depleted uranium-uranyl chloride on the mouse macrophage cell line, J774. Using a cytochemical staining protocol specific for uranium, we found that uranium uptake by the macrophages increased in a time-dependent manner. Treatment with 1, 10, or 100 microM depleted uranium-uranyl chloride resulted in decreased viability of the J774 cells within 24 h. Flow cytometric analysis of the treated cells with annexin V showed the translocation of phosphatidylserine from the inner face of the plasma membrane to the outer surface indicating the loss of phospholipid symmetry and the beginning of the apoptotic process. Significant differences in annexin V labeling between control cells and cells treated with 100 microM depleted uranium-uranyl chloride were apparent within 2 h. Other events associated with apoptosis, including morphological changes and DNA fragmentation, were also apparent after depleted uranium-uranyl chloride treatment. These results suggest that the uptake and concentration of soluble depleted uranium by macrophages initiates events that results in the apoptotic death of these cells.

New strategies for the prevention of radiation injury: possible implications for countering radiation hazards of long-term space travel.

New strategies for the prevention of radiation injuries are currently being explored with the ultimate aim of developing globally radioprotective, nontoxic pharmacologics. The prophylactic treatments under review encompass such diverse pharmacologic classes as novel immunomodulators, nutritional antioxidants, and cytokines. An immunomodulator that shows promise is 5-androstenediol (AED), a well-tolerated, long-acting androstene steroid with broad-spectrum radioprotective attributes that include not only protection against acute tissue injury, but also reduced susceptibility to infectious agents, as well as reduced rates of neoplastic transformation. Other potentially useful radioprotectants currently under study include the nutraceutical vitamin E and analogs, a chemically-engineered cytokine, interleukin-1beta, and a sustained-release formulation of an aminothiol, amifostine. Results suggest that a new paradigm is evolving for the prophylaxes of radiation injuries, based on use of newly identified, nontoxic, broad-spectrum prophylactic agents whose protective action may be leveraged by subsequent postexposure use of cytokines with organ-specific reparative functions.

Health effects of embedded depleted uranium.

The health effects of embedded fragments of depleted uranium (DU) are being investigated to determine whether current surgical fragment-removal policies are appropriate for this metal. The authors studied rodents implanted with DU pellets as well as cultured human cells exposed to DU compounds. Results indicate that uranium from implanted DU fragments distributes to tissues distant from implantation sites, including bone, kidney, muscle, and liver. Despite levels of uranium in kidney that would be nephrotoxic after acute exposure, no histological or functional kidney toxicity was observed with embedded DU, indicating that the kidney adapts when exposed chronically. Nonetheless, further studies of the long-term health impact are needed. DU is mutagenic and transforms human osteoblastic cells into a tumorigenic phenotype. It alters neurophysiological parameters in rat hippocampus, crosses the placental barrier, and enters fetal tissue. Preliminary data also indicate decreased rodent litter size when animals are bred 6 months or longer after DU implantation.

Effects of anesthesia and vehicle injection on circulating blood elements in C3H/HeN male mice.

The induction and maintenance of general anesthesia can lead to profound alterations of many organ systems, especially the cardiovascular, pulmonary, and nervous systems. Moreover, distinct from their cardiopulmonary effects, certain anesthetics can induce physiologic and behavioral changes, which may persist after recovery from anesthesia. Knowledge of the effects of anesthesia and anesthetic agents on hematologic measurements is important. Although the effects of anesthesia were clinically unapparent, the effect on levels of circulating blood elements was an important determinant for the results of our study. We sought to evaluate the effect of anesthesia and vehicle injection on the levels of circulating blood elements in C3H/HeN male mice. We used an automated hematology system to obtain complete blood counts with differentials in anesthetized and unanesthetized mice receiving subcutaneous injections of polyethylene glycol (PEG-400). Two days after a 30-min exposure to isoflurane anesthesia, mean white blood cell counts had deceased by 15.4%, mean neutrophil counts had decreased by 26.9%, and mean platelet counts by 11.2% compared with levels in unanesthetized mice. Our results indicate that the effect of anesthesia is an important consideration when circulating blood elements in mice must be measured.

Application of multivariate modeling for radiation injury assessment: a proof of concept.

Multivariate radiation injury estimation algorithms were formulated for estimating severe hematopoietic acute radiation syndrome (H-ARS) injury (i.e., response category three or RC3) in a rhesus monkey total-body irradiation (TBI) model. Classical CBC and serum chemistry blood parameters were examined prior to irradiation (d 0) and on d 7, 10, 14, 21, and 25 after irradiation involving 24 nonhuman primates (NHP) (Macaca mulatta) given 6.5-Gy (60)Co Υ-rays (0.4 Gy min(-1)) TBI. A correlation matrix was formulated with the RC3 severity level designated as the "dependent variable" and independent variables down selected based on their radioresponsiveness and relatively low multicollinearity using stepwise-linear regression analyses. Final candidate independent variables included CBC counts (absolute number of neutrophils, lymphocytes, and platelets) in formulating the "CBC" RC3 estimation algorithm. Additionally, the formulation of a diagnostic CBC and serum chemistry "CBC-SCHEM" RC3 algorithm expanded upon the CBC algorithm model with the addition of hematocrit and the serum enzyme levels of aspartate aminotransferase, creatine kinase, and lactate dehydrogenase. Both algorithms estimated RC3 with over 90% predictive power. Only the CBC-SCHEM RC3 algorithm, however, met the critical three assumptions of linear least squares demonstrating slightly greater precision for radiation injury estimation, but with significantly decreased prediction error indicating increased statistical robustness.

Delta-tocotrienol protects mice from radiation-induced gastrointestinal injury.

We recently demonstrated that natural delta-tocotrienol (DT3) significantly enhanced survival in total-body irradiated (TBI) mice, and protected mouse bone marrow cells from radiation-induced damage through Erk activation-associated mTOR survival pathways. Here, we further evaluated the effects and mechanisms of DT3 on survival of radiation-induced mouse acute gastrointestinal syndrome. DT3 (75-100 mg/kg) or vehicle was administered as a single subcutaneous injection to CD2F1 mice 24 h before 10-12 Gy (60)Co total-body irradiation at a dose rate of 0.6 Gy/min and survival was monitored. In a separate group of mice, jejunum sections were stained with hematoxylin and eosin and the surviving crypts in irradiated mice were counted. Apoptosis in intestinal epithelial cells was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and bacterial translocation from gut to heart, spleen and liver in irradiated mice were evaluated. DT3 (75 mg/kg) significantly enhanced survival in mice that received 10, 10.5, 11 or 12 Gy TBI. Administration of DT3 protected intestinal tissue, decreased apoptotic cells in jejunum and inhibited gut bacterial translocation in irradiated mice. Furthermore, DT3 significantly inhibited radiation-induced production of pro-inflammatory factors interleukin-1ß and -6 and suppressed expression of protein tyrosine kinase 6 (PTK6), a stress-induced kinase that promotes apoptosis in mouse intestinal cells. Our data demonstrate that administration of DT3 protected mice from radiation-induced gastrointestinal system damage.

Artifacts related to N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide derivatization of citrulline revealed by gas chromatography-mass spectrometry using both electron and chemical ionization.

Derivatization with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) was used for gas chromatography-mass spectrometry (GC-MS) analysis of citrulline and ornithine. Aqueous 50 microl aliquots at 1 and 10 mM concentrations were dried and derivatized separately, and 70 eV electron ionization or CH(4) positive chemical ionization were used. Ornithine produced a single GC peak. Physiological citrulline concentrations produced GC artifact peaks for the ornithine derivative, and a compound consistent with elimination of a water molecule from the tri-tert-butyldimethylsilyl (TBDMS) citrulline derivative. A third GC peak obtained using 10 mM citrulline concentrations gave a mass spectrum consistent with a mixture of true tri- and tetra-TBDMS citrulline. Analyses of (13)C-ureido-labeled citrulline confirmed the presence of the true TBDMS citrulline derivatives produced from 10 mM samples and provided evidence that the TBDMS ornithine artifact results from loss of TBDMS isocyanate from tetra-TBDMS citrulline. Linear-programmed temperature GC retention index data relative to n-alkanes are reported for observed GC peaks.

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.