Time‐course of micronucleated erythrocytes in response to whole‐body gamma irradiation in a model mammalian species,the bank vole (Clethrionomys glareolus,Schreber)

The time course of the formation of micronucleated polychromatic (MNPCEs) and normochromatic erythrocytes (MNNCEs) in the bone marrow of the bank vole (Clethrionomys glareolus, Schreber), a model mouse‐like species, was studied using the standard micronucleus test at 0, 6, 12, 18, 24, 30, 36 and 48 hr following whole‐body acute γ‐irradiation at a dose of 0.5 Gy. Based on the existing literature on laboratory mice, it was suggested that such a dose will not have significant effect on erythroid cell proliferation in the bank vole and hence on the time course of the rise of micronucleated cells. In total, ～905,000 polychromatic (PCEs) and normochromatic erythrocytes (NCEs) from 82 adult bank voles were analyzed. Although the mean frequencies of MNNCEs were too low to allow for the correct assessment of their time course, an analysis of PCEs showed an increasing rate of MNPCE appearance at 6 hr that reached a maximum at 18–24 hr after irradiation and subsequently decreased. Because the kinetics of MNPCEs reflects the process of erythropoiesis, the current results regarding the time points of appearance of radiation‐induced MNPCEs provide the first information on the prolongation of one of the terminal stages of erythrocyte formation in bank vole specimens, namely the stage of maturation of PCEs from erythroblasts. Moreover, the observed time‐course data, as well as the low‐background frequencies of MNPCEs and characteristic level of PCEs response to radiation, showed similarities between the two model species: bank vole (this study) and laboratory mice (literature data). Environ. Mol. Mutagen. 52:50–57, 2011. © 2010 Wiley‐Liss, Inc.     

Induction of cytogenetic damage in rodents after short-term inhalation of benzene

Experiments were designed to investigate both the induction of sister chromatid exchanges (SCEs) in peripheral blood lymphocytes (PBLs) and micronuclei (MN) in bone marrow polychromatic erythrocytes (PCEs) of mice and rats after inhalation of benzene (BZ). Male DBA/2 mice (17-19 weeks old) were exposed to target concentrations of either 0, 10, 100, or 1,000 ppm BZ for 6 hr. Male Sprague-Dawley rats (11-14 weeks old) were exposed to target concentrations of either 0, 0.1, 0.3, 1, 3, 10, or 30 ppm BZ for 6 hr. Blood was obtained by cardiac puncture 18 hr after exposure, and PBLs were cultured in the presence of lipopolysaccharide (mouse B cells, 60 micrograms/ml) or concanavalin A (rat T cells, 30 micrograms/ml) to stimulate blastogenesis for SCE analysis. Femoral bone marrow smears from both species were analyzed for MN in PCEs 18 hr after BZ exposure. Mouse PBLs revealed a significant concentration-related increase in the SCE frequency over controls at 10, 100, or 1,000 ppm BZ. Mouse bone marrow showed a significant concentration-dependent increase in MN over controls after exposure to 10, 100, or 1,000 ppm BZ. Rat PBLs showed a significant increase in the SCE frequency after exposure to 3, 10, or 30 ppm BZ. The statistical significance of the 1 ppm BZ result was borderline and dependent on the statistical test chosen. Rat cells revealed a significant concentration-related increase in MN after inhalation of either 1, 3, 10, or 30 ppm BZ. PBLs from treated mice showed significant concentration-dependent decreases in mitotic indices; however, cell cycle kinetics and leucocyte counts remained unaffected. Rat PBLs showed significant decreases in mitotic activity only after exposure to 3 and 30 ppm BZ, whereas cell cycle kinetics and leucocyte counts were unaffected. These results show that BZ can induce statistically significant cytogenetic effects in PBLs and PCEs of both mice and rats after a 6-hr inhalation of BZ at low concentrations.     

Effect of O6‐chloroethylguanine DNA lesions on the kinetics and mechanism of micronucleus induction in vivo

The aim of this work was to determine the kinetics of micronucleus production because of an increase in O⁶‐chloroethyl guanine (O6‐ChlEt‐G) DNA lesions in murine bone marrow cells in vivo. We increased the frequency of O6‐ChlEt‐G lesions by pretreatment with an inhibitor of O⁶‐methylguanine‐DNA methyltransferase (MGMT), O⁶‐benzylguanine (O6BG), and subsequent treatment with bis‐chloroethylnitrosourea (BCNU). The kinetics of micronucleated‐polychromatic erythrocyte (MN‐PCE) induction was established by scoring the frequency of MN‐PCEs per 2000 PCEs in peripheral blood at 8‐hr intervals from immediately prior to treatment to 72‐hr post‐treatment. We examined groups of five mice treated with (i) dimethylsulfoxide (DMSO), (ii) O6BG in DMSO, (iii) BCNU, or (iv) O6BG in DMSO plus BCNU. The data indicate that O6BG pretreatment causes: (i) ían increase in MN‐PCEs induced by BCNU, (ii) a delay in the time of maximal MN‐PCE induction produced by the different BCNU doses, and (iii) an increase in cytotoxicity. These data confirm that O6‐ChlEt‐G is a lesion involved in DNA break induction and in the subsequent production of micronuclei, and also that these lesions seem to be stoichiometrically reduced by MGMT. These data also show that induction of MN‐PCEs by BCNU is delayed by pretreatment with O6BG for more than 6 hr, perhaps due to the time required for repair of crosslinks derived from O6‐ChlEt‐G and/or for DNA duplication, which is required for adduct transformation into crosslinks. Environ. Mol. Mutagen. 2010. © 2009 Wiley‐Liss, Inc.     

Repeated exposures to gustatory stimuli produce habituation or positive contrast effects in perinatal rats

Adult rats exhibit a decrease in consummatory responses following repeated presentations of a taste (habituation) and an increase in consummatory responses if they experience an upward shift in the magnitude or intensity of a gustatory stimulus (e.g., sucrose or saccharin). These responses do not represent a direct sensorimotor reaction to a gustatory cue, but rather reflect a change in responding based on the memory of a previous taste. Here, we sought to determine if fetal rats could (like adults) adjust their orofacial motor responses based on a memory of recent gustatory experience. Embryonic Day 18 (E18) or Day 19 (E19) rat fetuses received oral lavage with either 0.15 or 0.30% saccharin (SAC). Subsequently, observations of orofacial movements (mouthing and licking) following oral lavage with 0.30% SAC were made 50 min later, 24 hr later, or on postnatal Day 3 (P3). Thus, some animals were in a "shifted" condition in which they first experienced a relatively low concentration of SAC and then a higher one while control rats ("nonshifted") received 0.30% SAC during both taste exposures. Fetuses exhibited evidence of both habituation (with repeated presentation of the 0.30% SAC) and positive contrast effects (PCEs) (following an upward shift in SAC concentration) when retested 50 min after their first exposure to SAC on E19. However, these animals did not exhibit PCEs 24 hr later or 5 days later (on P3). Contrast effects were not observed when the initial SAC exposure was on E18, and habituation responses were variable depending on the time interval between the taste presentations to these animals. Rats with a 5- to 6-day latency between the two taste presentations showed neither PCEs nor habituation. Our data indicate that PCEs and habituation effects emerge at different ages, and their demonstration is dependent upon the latency between the taste presentations.     

DNA content determination of micronucleated polychromatic erythrocytes induced by clastogens and spindle poisons in mouse bone marrow and peripheral blood

The frequencies and DNA distributions of micronuclei (MN) inpolychromatic erthrocytes (PCEs) from bone marrow (BM) and peripheralblood (PB) of mice after four different treatments were determinedby flow cytometry. PCEs were differentiated from normochromaticerythrocytes (NCEs) using the fluorescent RNA stain Thiazoleorange, while MN in erythrocytes were detected with the DNAstain Hoechst 33342. The treatments were X-irradiation (1 Gy)cyclophosphamide (CPA; 30 mg/kg) vincristine sulphate (VCR;0.08 mg/kg) and cholchicine (COL; 1 mg/kg). All treatments showedincreased frequencies of micronucleated PCEs at 30 h after treatmenthi BM and at 50 h in PB. The clastogens(X-irradiation and CPA)and the spindle poisons (VCR and COL) could be grouped accordingto the fluorescent characteristics of the induced MN as wellas the relative frequency of small (0.5—2% of the diploidDNA content) and large (2—10%) MN. In PB the relativefrequency of large MN was lower than in BM indicating that theywere partly eliminated before entrance into the peripheral circulation. ³To whom correspondence should be addressed     

An ultrastructural study of the influence of radiation against cell line-derived human oral malignant melanoma

The response to radiation of the SSM-1 cell line, derived from oral malignant melanoma, was investigated by survival curve and by light and electron microscopic abservation. Survival curve of the cells was investigated by the colony formation assay. X-irradiation with 2, 4, and 8 Gy was performed against the cell line. Morphological changes of the cells at 6, 24, 72, and 120h after irradiation were examined by both light and electron microscopy. The survival curve of the SSM-1 cells showed higher radiosensitivity than that of cutaneous melanoma cell lines. Giant cells and multinucleated cells were found only 120h after 8Gy irradiation. Ultrastructural observation revealed changes in nucleus and melanosomes; melanosomes increased in number at 120h after 8Gy irradiation. Further alterations after irradiation were noticed primarily in the nucleus. Radiosensitivity of the SSM-1 cell line derived from oral malignant melanoma was higher than that of cutaneous melanoma cell lines. The results of this study may support the concept that radiotherapy is effective for oral malignant melanoma.This study was presented at the 27th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Kurashiki, September 28–30, 1995.     

γH2AX formation kinetics in PBMCs of rabbits exposed to acute and fractionated radiation and attenuation of focus frequency through preadministration of a combination of podophyllotoxin and rutin hydrate

DNA damage can be assessed by the quantitation of γH2AX foci that form at DSB sites. This study examines the generation and persistence of γH2AX foci, variability in foci size after acute and fractionated radiation exposure, and the effect of pretreatment with a safe radioprotective formulation termed G‐003M on foci generation and persistence. G‐003M contains a combination of podophyllotoxin and rutin hydrate, and was administered intramuscularly to rabbits 1 hr prior to Co⁶⁰ gamma irradiation. Rabbits were assigned to one of the following treatment groups: untreated, G‐003M alone, irradiated (single dose 8 Gy, fractionated 2 Gy/day for 4 days or single dose 2 Gy) or G‐003M preadministration followed by radiation exposure. Foci continuously persisted for a week in peripheral blood mononuclear cells of rabbits exposed to a single 8 Gy dose. However, the number of foci gradually decreased after reaching a maximum at 1 h. In rabbits exposed to fractionated radiation, foci detected 1 hr after the final exposure were significantly larger (P < 0.001) than in rabbits exposed to a single 8 Gy dose, but disappeared completely after 24 h. In both groups, foci reappeared on days 11‐15 in terminally ill animals. G‐003M pretreatment significantly (P < 0.05) attenuated the formation of γH2AX foci in all irradiated rabbits. This study reveals that γH2AX focus assessment could be used to confirm radiation exposure, that focus size reflects the type of radiation exposure (acute or fractionated), that the re‐appearance of foci is a strong indicator of imminent death in animals, and that G‐003M provides protection against radiation. Environ. Mol. Mutagen. 57:455–468, 2016. © 2016 Wiley Periodicals, Inc.     

IL-2对辐射损伤后小肠上皮细胞生长影响的实验研究

目的: 观察电离辐射对体外培养的IEC -6细胞株生长的影响及IL- 2对其损伤后增殖和恢复的作用, 并进一步探讨肠黏膜免疫与肠上皮辐射损伤及修复的关系。方法: 用 4、8、12Gy的γ射线照射IEC- 6细胞株, 并于照后 3、6、9、12h及 1、2、3d, 用MTT比色法、光镜、电镜、DNA凝胶电泳和流式细胞术等, 检测受照射后IEC- 6细胞的增殖活力、形态和死亡方式的改变; 用不同浓度IL 2 ( 25×103、5×104、1×105U/L)处理 8Gyγ射线照射的IEC 6细胞, 并于照射后 3、6、9、12、24h, 采用MTT比色法检测其增殖活力的变化。结果: 在 0~12Gy的范围内, IEC 6细胞的增殖活力随γ射线照射剂量的增加而降低。8. 0γ射线照后 24h, 凋亡的IEC- 6细胞明显增多, DNA凝胶电泳显示有梯状带形成。IL- 2可促进照射后的IEC- 6细胞增殖且呈一定的剂量 效应关系, 尤以1×105U/L组的作用更明显。结论: 在一定剂量范围内, γ射线照射可降低IEC 6细胞增殖活力, 且存在剂量 效应关系;可导致IEC 6细胞发生凋亡。IL- 2可促进受照射的IEC- 6细胞增殖, 增强其抗辐射的作用。     

Radiotherapy in the management of Kaposi's sarcoma: comparison of 8 Gy versus 6 Gy

OBJECTIVE: To evaluate prospectively the efficacy of a single fraction of high-dose radiotherapy in patients with Kaposi's sarcoma. PATIENTS: Between 1994 and 2004, 47 patients with Kaposi's sarcoma were treated at Hacettepe University, Department of Radiation Oncology. Thirteen (28%) patients received chemotherapy before radiotherapy and were referred due to recurrent or progressive disease or intolerance to chemotherapy. All lesions were treated locally with a 2-3-cm safety margin with 4-6-MeV electron beams. Radiotherapy consisted of a single fraction of 8 Gy in the first four years and 6 Gy thereafter. RESULTS: The male:female ratio was 4:1. The median age was 61 years (range 18-87). Eight out of 47 patients (17%) had an underlying immunocompromised state, and one had a previous diagnosis of Hodgkin's disease. Of 203 fields treated, 51 and 152 fields were treated with 8 Gy and 6 Gy, respectively. Overall response rates (RR) at 12 months for 8- and 6 Gy were 93% and 86%, which were not statistically different. However, the difference between complete RRs at 12 months (93% and 60% for 8 Gy and 6 Gy respectively) was significant (p<0.0001). Progression-free survival and reirradiation rates were not significantly different. Side effects were tolerable in all but three patients with grade 2-3 fibrosis and edema. CONCLUSION: Radiotherapy is an effective mode of treatment for Kaposi's sarcoma, and a single dose of 8 Gy is more effective in terms of complete RR compared to 6 Gy, though overall response and progression-free survival rates were similar.     

Short exposures to enriched environments can increase genetic variability of behavior in mice.

Previous work indicates that mice of different genotypes reared in enriched environments show differential increases in performance on a food-seeking task. In this study 2 experiments examined the effects in selected mice strains of short exposures to such enrichment. Experiment 1 indicated that 48 hr of exposure to enriched cages was sufficient to produce results found previously when subjects were reared from birth in enriched cages. Experiment 2 indicated that as little as 6 hr of exposure to an enriched cage was sufficient to produce almost maximal enrichment effects in C57BL/10J mice.     

Detection of cytomegalovirus-infected cells by flow cytometry and fluorescence in suspension hybridisation (FLASH) using DNA probes labeled with biotin by polymerase chain reaction.

A biotin-labeled DNA probe specific for the immediate early gene of the cytomegalovirus (CMV) strain AD 169 was generated with the polymerase chain reaction. Nucleic acid hybridisation was carried out with CMV-infected T-lymphoblastoid cells (MOLT-4) after 4 hr to 6 days of culture. Biotin molecules were made visible with streptavidin coupled with fluorescein. The fluorescence signal of the hybridised probe was measured by flow cytometry in the cell suspension. The number of CMV-positive cells was 7% at 4 hr, 8% after 28 hr, 18% after 2 days, 26% after 3 days, 91% after 4 days, 97% after 5 days, and 98% after 6 days. The first detection of CMV antigen (pp65) was possible with immunoenzymatic labeling by day 4, whereas CMV-DNA was detected by PCR after 4 hr. CMV-specific RNA could be detected in a similar way. The analysis of mononuclear peripheral blood leukocytes in a patient with active CMV infection showed 14.7% CMV DNA-positive cells at day 1 and 7% at day 8, as compared to 0.9% and 0.0% cells which were positive for CMV antigen (pp65) by immunoenzymatic labeling at day 1 and day 8, respectively. We conclude that flow cytometry and fluorescence in suspension hybridisation (FLASH) offers a new tool for analysing exactly and quantifying large numbers of cells for specific DNA or RNA and may be useful for other laboratory and clinical applications.     

The physiological rhythms of subjects living on a day of abnormal length.

1. Fourteen subjects, singly or in groups, have been observed while living on a 21 hr day for 8 or 16 experimental 'days' and fifteen other subjects similarly on a 27 hr day. 2. Rhythmic components of body temperature and excretion of various urinary constituents were calculated. 3. On a 21 hr day, for most components and most subjects, two periods were present, one of 21 hr and one of around or somewhat over 24 hr. 4. On a 27 hr day two periods were less often present and a larger number of observed rhythms could be satisfactorily described by a single period, usually between 23 and 28 hr. 5. In subjects spending a second week on a 21 hr day the circadian component was no less prominent than during the first week. 6. When, after life on a 21 hr day, subjects were deprived of knowledge of time, there was evidence that the 21 hr component did not persist. 7. The results are interpreted as evidence of the continuing existance of an influence with a period of around 24 hr, simultaneously rhythmic influences resulting from the subjects' habits. On a 27 hr day there was sometimes evidence of entrainment, yielding an intermediate period. 8. An attempt is made to compare the relative potency of the exogenous and of the persistent circadian influences on the several variables.     

Alterations in gene expression profiles and the DNA-damage response in ionizing radiation-exposed TK6 cells

Identifying genes that are differentially expressed in response to DNA damage may help elucidate markers for genetic damage and provide insight into the cellular responses to specific genotoxic agents. We utilized cDNA microarrays to develop gene expression profiles for ionizing radiation-exposed human lymphoblastoid TK6 cells. In order to relate changes in the expression profiles to biological responses, the effects of ionizing radiation on cell viability, cloning efficiency, and micronucleus formation were measured. TK6 cells were exposed to 0.5, 1, 5, 10, and 20 Gy ionizing radiation and cultured for 4 or 24 hr. A significant (P < 0.0001) decrease in cloning efficiency was observed at all doses at 4 and 24 hr after exposure. Flow cytometry revealed significant decreases in cell viability at 24 hr in cells exposed to 5 (P < 0.001), 10 (P < 0.0001), and 20 Gy (P < 0.0001). An increase in micronucleus frequency occurred at both 4 and 24 hr at 0.5 and 1 Gy; however, insufficient binucleated cells were present for analysis at the higher doses. Gene expression profiles were developed from mRNA isolated from cells exposed to 5, 10, and 20 Gy using a 350 gene human cDNA array platform. Overall, more genes were differentially expressed at 24-hr than at the 4-hr time point. The genes upregulated (> 1.5-fold) or downregulated (< 0.67-fold) at 4 hr were those primarily involved in the cessation of the cell cycle, cellular detoxification pathways, DNA repair, and apoptosis. At 24 hr, glutathione-associated genes were induced in addition to genes involved in apoptosis. Genes involved in cell cycle progression and mitosis were downregulated at 24 hr. Real-time quantitative PCR was used to confirm the microarray results and to evaluate expression levels of selected genes at the low doses (0.5 and 1.0 Gy). The expression profiles reflect the cellular and molecular responses to ionizing radiation related to the recognition of DNA damage, a halt in progression through the cell cycle, activation of DNA-repair pathways, and the promotion of apoptosis.     

Superantigen activation and kinetics of cytokines in the Long-Evans rat.

This study was conducted to identify and quantify, over time, selected cytokine responses in Long-Evans rats that were exposed to staphylococcus enterotoxin B (SEB). The kinetics of selected cytokines [interleukin-2 (IL-2), IL-6, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF)] and phenotype and cell cycle analysis of T lymphocytes were determined in Long-Evans rats administered a single intraperitoneal (i.p.) dose of either 50 microg or 500 microg of SEB. Rats injected with 50 microg SEB had significantly elevated levels of IL-2, IL-6 and IFN-gamma in their serum 2 hr post-injection. IL-2 serum levels were significantly elevated at 2 hr and returned to near control values by 12 hr while both IL-6 and IFN-gamma peaked at 6 hr but remained significantly increased at 24 hr post SEB exposure. A 500 microg dose of SEB did not further enhance these cytokine responses. When spleen cells were collected for culture 2 hr after rats were injected i.p. with 50 microg SEB and cocultured with SEB, TNF and IL-6 levels were significantly increased after 2 hr incubation, while IL-2 and IL-6 were significantly elevated at 6 hr. Production of all these cytokines in spleen cell cultures continued to increase over the 24 hr sampled. Peritoneal cells were collected for culture either at 1 hr or 2 hr after injection of either 50 microg or 500 microg of SEB. IL-6 was significantly increased after 1 hr in culture while TNF was significantly increased by 2 hr regardless of whether the cells were harvested 1 or 2 hr after SEB injection. The greatest response for both IL-6 and TNF occurred when cells from animals injected with 50 microg SEB were restimulated in vitro with SEB. The peak levels for IL-6 were at 12 hr post SEB exposure while TNF peaked at 6 hr. The percentage of CD4+ cells was significantly increased at 48 hr and 72 hr post SEB (50 microg) administration while the percentage of CD8+ cells remained similar to control values for the 168-hr test period. A similar pattern was observed in cell cycling where the CD4+ cells proliferated up to 2 days post SEB injection and then were significantly suppressed at day 3. The CD8+ cells were comparable to control values. These studies demonstrate that the cytokine responses in Long-Evans rats exposed to a superantigen are somewhat similar to those that occur in mice and humans, e.g. a rapid short increase in the production of IFN-gamma and TNF that was accompanied by an increase in the production of IL-2. Additional responses noted in this species, however, were a marked increase in IL-6 production, as well as an early increase in the number and cycling of CD4+ cells followed by a down-regulation of these events. These activities occurred in the absence of notable histopathological alteration of lymphoid organs. The results indicate that the Long-Evans rat is an acceptable animal model to investigate the pathogenesis of superantigen-induced disease and that IL-6 may be an active mediator of this process.     

Long-term habituation to repeated loud noise is impaired by relatively short interstressor intervals in rats

The phenomenon of spaced (longer intertrial interval) compared with massed (shorter intertrial interval) training leading to better long-term habituation and associative learning is well documented. However, the effects of intertrial intervals on response habituation to repeated stress exposures have not been previously examined. The present experiments found that massed (six 30-min exposures of 95 dB white noise in 6 hr) and spaced (one 30-min exposure daily for 6 days) noise exposures led to similar habituation of plasma corticosterone and ACTH responses, heart rate, and core body temperature after the 6th exposure in male Sprague-Dawley rats. However, these habituated responses were not retained in the massed group on a similar noise re-exposure 48 hr later, compared with the spaced group. The habituated responses found in the massed group after the 6 noise exposures were not due to differential hearing threshold shifts, as examined with modifications of the acoustic startle reflex. These data indicate that relatively short interstressor intervals impair long-term stress adaptation. This series of studies supports the idea of distinct short- and long-term habituation processes to stress responsiveness.     

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.     

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: 17α-androstenediol, dehydroepiandrosterone, 5-androstenetriol (AET), 4-androstenedione (AND), testosterone, estradiol, fluasterone, 16α-bromoepiandrosterone, 16α-fluoro-androst-5-en-17α-ol (α-fluorohydrin, AFH), and 16α-fluoro-androst-5-en-17β-ol (β-fluorohydrin). Steroids were administered 24 or 48 hr before, or 1 hr after, whole-body γ-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 (sc) injection of 5-AED is as long as 48 hr in mice. Moreover, the results indicate that 1 sc 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 sc injection of extremely similar molecules with the same physicochemical properties as 5-AED were not radioprotective; 2) the17-hydroxyl group is essential; 3) this group must be in the β configuration in the absence of nearby side groups; 4) a halogen atom at 16 changes the 17-hydroxyl specificity to α; 5) the 3β-hydroxyl group is not essential; 6) addition of a 7β-hydroxyl group is deleterious; and 7) the effects are not due to activation of sex steroid receptors.     

Uptake of tritiated leucine by axotomized cervical motoneurons: An autoradiographic study

Kittens, aged 7–9 weeks, were killed in groups of four at survival periods of 0 (unoperated controls), 1, 2, 3, 5, 10, 15, and 28 days following left brachial plexotomy. [³H]Leucine was administered intraperitoneally, 10 μCi/g body wt, 0.25, 0.5, 2, or 16 hr before sacrifice. Qualitative and quantitative histologic and autoradiographic observations were made. On the axotomized side, chromatolysis was qualitatively evident in 97% of C-8 motoneurons 10 days postoperatively. Cellular enlargement was highly variable. Nuclear atrophy was frequent 10–15 days after surgery. Regenerating axons penetrated the neuroma 5 days after plexotomy and reached the ulnar nerve at the level of the olecranon at 28 days and neuromuscular junctions of flexor carpi ulnaris at 60 days. Axotomized cervical motoneurons from kittens sacrificed 0.25 hr after administration of [³H]leucine on the 10th or 15th postoperative day exhibited a relative decrease in the uptake of labeled amino acid. A similar decrease occurred in the 28-day survival sacrificed 0.50 hr after injection of labeled amino acid. In contrast when animals were killed at the 2.0 hr postinjection period, a relative increase in the somal accumulation of tritiated leucine occurred 2 through 28 days post-operatively. A comparable increase obtained 16.0 hr after isotope injection and 2–10 days after plexotomy. The differences in the direction of change observed at short (0.25 and 0.5 hr) and long (2.0 and 16.0 hr) labeling intervals suggest that alterations in accumulation of radiolabeled amino acid by axotomized somas may be related to changes in the types of proteins synthesized or to alterations in the rates of export and/or turnover of cytoplasmic proteins. The increased cytoplasmic radioactivity observed after 2.0- and 16.0-hr labeling intervals preceded light microscopic evidence of axon regeneration and was not dependent on reestablishment of peripheral contacts.     

The secondary immune response to Staphylococcus aureus vaccines in efferent popliteal lymph of sheep.

The secondary immune response to live and killed Staphylococcus aureus vaccines was studied in efferent popliteal lymph of sheep. Animals were immunized with either live or killed S. aureus intracutaneously on the lateral hock, in an area draining into the popliteal lymph node. Six weeks later, an efferent popliteal lymphatic vessel in the vaccinated leg was cannulated, and 48 hr after surgery a second inoculation (identical to the primary) was placed in the skin adjacent to the primary vaccination lesion. A dramatic decrease in lymphocyte output ('cell shutdown') was observed in lymph collected from sheep given the secondary inoculation of live S. aureus during the first 8 hr after inoculation. However, only a moderate decrease in lymphocyte output occurred in lymph from animals receiving killed S. aureus or from control animals. The proportion of eosinophils in lymph collected from animals given live S. aureus increased to a peak (14% of total leucocytes in lymph) between 6 hr and 8 hr, and returned to prechallenge levels by 24 hr post-inoculation. The percentage of neutrophils in lymph peaked between 8 hr and 1 day after injection of live bacteria. This granulocyte response was not observed in animals given killed S. aureus or control animals. IgM-, IgG1- and IgG2- containing cells (-cc) in lymph were quantified by indirect immunofluorescence. Animals given live S. aureus produced lymph with greater numbers of Ig-cc of these isotypes than those given killed organisms. The ratio of IgG2-cc:IgG1-cc was significantly greater in lymph from animals given live S. aureus from Day 2 to Day 6 post-challenge. IgM and IgG1 anti-staphylococcal antibody levels increased in lymph collected from all vaccinated animals, but only sheep given live S. aureus showed any increase in levels of IgG2 antibody.     

Fever alters characteristics of sleep in rats

This study examined the effects of a fungal infection on body temperature (Tb) and sleep states. Tb and sleep were recorded in male rats for 24 hr after a saline injection and 48 hr after a subcutaneous injection of live brewer's yeast, at ambient temperatures (Ta's) of 20 degrees and 30 degrees C. Peak fevers of 1.6-3.1 degrees C occurred within 6-10 hr at both Ta's. The rats remained febrile for the next 12-24 hr. For the first 24 hr postyeast, amounts of SWS increased by 19 +/- 3% at 20 degrees C and 12 +/- 2% at 30 degrees C. Specifically, SWS was significantly increased from hr 5-8 (lights-on) and 13-24 (lights-off) at 20 degrees, and from hr 5-8 and 17-24 at 30 degrees C. Ta did not affect the changes in Tb or the changes in SWS after either saline or yeast. Duration of REMS varied with Ta after saline. After yeast, REMS increased by 21 +/- 12% at 20 degrees and decreased by 28 +/- 6% at 30 degrees C, with the net result that REMS at the two Ta's was equal during the fever. Furthermore, while the rats were febrile the normal diurnal variation in REMS was eliminated. Sleep and Tb returned to control values during the second fever day. These results suggest that an activated immune system both increases SWS and overrides the diurnal and thermoregulatory modulations of REMS.