Relieved residual damage in the hematopoietic system of mice rescued by radiation-induced adaptive response (Yonezawa Effect)

Existence of adaptive response (AR) was previously demonstrated in C57BL/6J mice. Irradiations were performed by delivering a priming low dose of X-rays (0.50 Gy) in combination with a challenge high dose of accelerated carbon or neon ion particles. AR was characterized by significantly decreased mortality in the 30-day survival test. This mouse AR model (‘Yonezawa Effect’) was originally established by using X-rays as both the priming and challenge irradiations. The underlying mechanism was due to radio-resistance occurring in blood-forming tissues. In this study, we verified the existence of AR and further investigated residual damage in the hematopoietic system in surviving animals. Results showed that the priming low dose of X-rays could relieve the detrimental effects on the hematopoietic system. We observed both an improvement in the blood platelet count and the ratio of polychromatic erythrocytes (PCEs) to the sum of PCEs and normochromatic erythrocytes (NCEs) and a marked reduction of the incidences of micronucleated PCEs and micronucleated NCEs. These findings suggest that the priming low dose of low linear energy transfer (LET) X-rays induced a protective effect on the hematopoietic system, which may play an important role in both rescue from acute lethal damage (mouse killing) and prevention of late detrimental consequences (residual anhematopoiesis and delayed genotoxic effects) caused by exposure to a high challenge dose from low-LET (X-ray) or high-LET (carbon and neon ion) irradiations. These findings provide new knowledge of the characterization of the Yonezawa Effect by providing new insight into the mechanistic study of AR in vivo.     

烟、茶对放射线所致骨髓细胞突变和骨髓抑制的影响

目的 探讨烟、茶对放射线所致小鼠骨髓细胞突变和骨髓抑制的干预作用,为放射损伤的防护提供基础资料。方法 在7组动物中分别给予吸烟、饮茶和放射线以及三因素的不同组合,另2组分别为空白对照组和环磷酰胺阳性对照组。测定小鼠骨髓嗜多染红细胞(PCE)微核率,同时观察PCE与成熟红细胞(RBC)的比例,进行外周血白细胞(WBC)计数。结果 被照射的各组PCE微核率均高于空白对照组(P <0.05或0.01),吸烟+放射组PCE微核率高于放射组(P <0.05)。被照射的各组PCE/RBC比值均低于空白对照组(P <0.01),被照射的各组WBC计数均低于空白对照组(P <0.05或0.01)。结论 放射线具有致突变作用,吸烟可增强放射线的致突变作用,饮茶不能抑制放射线的致突变作用,饮茶也不能抑制吸烟增强放射线的致突变作用;放射线可引起骨髓抑制,烟、茶对放射线引起的骨髓抑制无明显影响。     

Amelioration of radiation-induced hematopoietic and gastrointestinal damage by Ex-RAD(R) in mice

The aim of the present study was to assess recovery from hematopoietic and gastrointestinal damage by Ex-RAD^®, also known as ON01210.Na (4-carboxystyryl-4-chlorobenzylsulfone, sodium salt), after total body radiation. In our previous study, we reported that Ex-RAD, a small-molecule radioprotectant, enhances survival of mice exposed to gamma radiation, and prevents radiation-induced apoptosis as measured by the inhibition of radiation-induced protein 53 (p53) expression in cultured cells. We have expanded this study to determine best effective dose, dose-reduction factor (DRF), hematological and gastrointestinal protection, and in vivo inhibition of p53 signaling. A total of 500 mg/kg of Ex-RAD administered at 24 h and 15 min before radiation resulted in a DRF of 1.16. Ex-RAD ameliorated radiation-induced hematopoietic damage as monitored by the accelerated recovery of peripheral blood cells, and protection of granulocyte macrophage colony-forming units (GM-CFU) in bone marrow. Western blot analysis on spleen indicated that Ex-RAD treatment inhibited p53 phosphorylation. Ex-RAD treatment reduces terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay (TUNEL)-positive cells in jejunum compared with vehicle-treated mice after radiation injury. Finally, Ex-RAD preserved intestinal crypt cells compared with the vehicle control at 13 and 14 Gy. The results demonstrated that Ex-RAD ameliorates radiation-induced peripheral blood cell depletion, promotes bone marrow recovery, reduces p53 signaling in spleen and protects intestine from radiation injury.     

Dietary supplementation with annatto food-coloring extracts increases the resistance of human erythrocytes to hemolysis

Erythrocytes exhibit high susceptibility to hemolysis in several pathologies due to the oxidation of cellular components. We hypothesized that annatto carotenoids improve the redox status of erythrocyte plasma membranes and promote a consequent increase in human erythrocyte resistance to hemolysis. The objective of this study was to evaluate whether food-grade annatto carotenoids can increase human erythrocyte resistance to hemolysis in vitro and ex vivo. For the in vitro experiment, erythrocytes from healthy volunteers were isolated and coincubated with bixin (BIX) or norbixin (NBIX) and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), glucose, or sodium nitrite (NaNO₂) as hemolysis inducers. In the ex vivo study, healthy volunteers consumed a capsule containing BIX or NBIX (0.05 mg/kg body weight per day) or placebo for 7 days before blood sample collection. Their erythrocytes were isolated and incubated with AAPH, glucose, or NaNO₂. In both the ex vivo and in vitro studies, erythrocytes were subjected to osmotic fragility tests. The activity of antioxidant enzymes, and reduced glutathione and lipid peroxidation levels in erythrocytes were also evaluated ex vivo. In vitro BIX and NBIX not only reduced erythrocyte membrane fragility induced by AAPH, glucose, or NaNO₂ but also improved basal osmotic resistance in the micromole-per-liter range (P < .05). BIX and NBIX supplementation increased erythrocyte membrane resistance (P < .05), with BIX being more effective. Also, BIX and NBIX protected erythrocytes from lipid peroxidation and improved the cellular redox environment (P < .05). These results support the hypothesis that annatto carotenoids supplementation exerts antihemolytic properties by preventing the oxidative damage of human erythrocytes.     

Chloropentafluorobenzene: short-term inhalation toxicity, genotoxicity and physiologically-based pharmacokinetic model development.

Ten Fischer 344 rats and six B6C3F1 mice of each sex were exposed to air, 0.25, 0.80, or 2.50 mg chloropentafluorobenzene (CPFB)/liter of air for three weeks, excluding weekends. Exposure to 2.50 mg/liter caused a reduction in the growth rate of rats but did not affect the growth rate of mice. Following the exposure there was reduced SGOT activity in the blood serum of exposed rats and a dose related increase in liver weights. Increased liver weights were observed in mice as well; the response in the female groups was clearly dose dependent. Histologically the livers of both rats and mice presented single cell necrosis. In exposed mice hepatocytes exhibited mild hepatocytomegaly with increased granular eosinophilic cytoplasm. In evaluations for its potential to induce chromosomal damage following this exposure regimen, CPFB did not alter the rate of bone marrow cellular proliferation. Assessment of the micronucleated polychromatic erythrocytes and normochromatic erythrocyte populations during the inhalation exposures indicated a general absence of genotoxic activity.     

Recent progress in the development of malaria vaccines: Memorandum from a WHO Meeting

The sixth meeting of the Scientific Working Group on the Immunology of Malaria reviewed studies on the identification and analysis of malarial antigens of asexual blood stages and sexual stages (gametes, zygotes, ookinetes) that may be exploited as targets for vaccination. Several proteins have been identified on the surface of mature schizonts and free merozoites, some of which can be recognized by antibodies which block in vitro parasite growth. Immunization of rodents and monkeys with purified antigens from the parasite surface membrane has conferred substantial immunity against subsequent challenge. A new class of malarial antigens has been identified which bind specifically to glycophorin, the major erythrocyte glycoprotein; these antigens are on the merozoite surface and it is possible that they mediate attachment to erythrocytes. Antibodies against these proteins also block parasite growth in vitro. The Plasmodium falciparum S-antigens have been characterized biochemically and the genes for two of these proteins sequenced. Several antigens have been localized in the invasion process, and monoclonal antibodies against these proteins block in vitro growth. Malarial antigens on the surface of P. falciparum trophozoite and schizont-infected erythrocytes may be involved in the cytoadherence of infected erythrocytes to endothelial cells. Surface antigens on gametes and zygotes of P. gallinaceum and P. falciparum have been shown to be the targets of transmission-blocking immunity. Monoclonal antibodies specific for these antigens block fertilization in the mosquito midgut. Transmission of P. gallinaceum can also be blocked by an antibody that blocks development of zygotes into ookinetes. Studies on the transmission of P. yoelii have identified a gamete protein that immunizes mice against transmission to mosquitos.     

Significant calcium accumulation in fragile erythrocytes by dietary restriction in mice

Previously we reported that mice adapted to a 40% dietary restriction showed shortened erythrocyte life span (Internat. J. Vit. Nutr. Res. 1989, 59, 406-412). To clarify the mechanism of this phenomenon, influences of dietary restriction on erythrocyte fragility and on some factors associated with erythrocyte deformability were investigated. Forty percent dietary restriction caused an increase of erythrocyte osmotic fragility, a remarkable decrease of ATP levels and a striking accumulation of calcium in erythrocytes. In the diet restricted mice, it was further observed in the erythrocytes that the activity of rate limiting enzymes of glycolysis was significantly decreased. Remarkable decreases of the activities of calmodulin and membrane Ca2+/Mg2(+)-ATPase were also demonstrated in the erythrocytes from the diet restricted mice. These results strongly suggest that the fragile and short-lived erythrocytes in the diet restricted mice may be caused by the decreased activity of pumping out calcium from the cells. The fragile erythrocytes may be destroyed and removed from the blood flow earlier than flexible erythrocytes of the control mice. However, it is also suggested that the essential function of the erythrocytes, the oxygen supply to the peripheral tissues, may be maintained as 2,3-bisphosphoglyceric acid levels were not altered with erythrocyte aging in spite of the decreased glycolytic activity in the diet restricted mice.     

The acute toxic effects of copper on the blood of a teleost

Blood dyscrasias attributable to copper poisoning were observed in Colisa fasciatus, a freshwater teleost, 90 hr after exposure to 3 mg/liter of copper nitrate; the 96-hr LC₅₀ values for the metal was 4 mg/liter. Total erythrocyte count, hematocrit, hemoglobin, and numbers of circulating mature erythrocytes and thrombocytes (per thousand cells of all types) increased significantly, but there were significant decreases in the number of small and large lymphocytes; blood clotting time and erythrocyte sedimentation rate also declined markedly in fish exposed to the metal. Total leukocyte and thrombocyte counts, number of immature red blood cells, and hepatosomatic index did not differ between experimental and control fish. The data obtained in this study are compared with previously reported observations on C. fasciatus and other teleosts exposed to various heavy metals.     

Mutagenicity of various organic fractions of diesel exhaust particles

Treated with various organic fractions of Diesel Exhaust Particles (DEP), the Ames test withSalmonella typhimurium strains TA98 and TA100, and the mice micronucleus test were employed to study the mutagenic activity in the bacterial reverse mutation system, with and without a mammalian S₉ activation component, and the clastogenic activity in mice polychromatic erythrocyte (PCE) stem cells. Extracted ultrasonically with dichloromethane then using the acid and base separated reaction and column chromatography, DEP were divided into five organic fractions. They are the organic acid fraction (Fl), the organic base fraction (F2), the aliphatic hydrocarbon fraction (F3), the aromatic hydrocarbon fraction (F4) and the polar fraction (F5). Results showed that an increase in the counted numbers of histidine revertants on theSalmonella TA100 and TA98 was observed with or without (S₉ mix), but these activities were more pronounced in the TA98 strains especially in the absence of the S₉ mix. These results suggest that the organic fractions of DEP contain mainly compounds with direct frame-shift mutaganicity. Positive results were also obtained from mice micronucleus assay. The frequency of mice bone marrow micronucleated polychromatic erythrocytes (PCE) was increased using this assay and it showed a definite dose-response relationship. The results suggest that various organic fractions could affect spindle fiber function or formation in mammalian cells. Compared with the results of different organic fraction, the effects of the F2, F4 and F5 were found to be stronger than those of other fractions. Based on the findings obtaind in the Ames and micronucleus tests, DEPs have genotoxic effects in both of the test systems. The project was supported by the National Nature Science Foundation of China, No 39270576     

Radioprotective effects of hawthorn fruit extract against gamma irradiation in mouse bone marrow cells

The radioprotective effect of hawthorn (Crataegus microphylla) fruit extract against genotoxicity induced by gamma irradiation has been investigated in mouse bone marrow cells. A single intraperitoneal (ip) administration of hawthorn extract at doses of 25, 50, 100 and 200 mg/kg 1h prior to gamma irradiation (2 Gy) reduced the frequencies of micronucleated polychromatic erythrocytes (MnPCEs). All four doses of hawthorn extract significantly reduced the frequencies of MnPCEs and increased the PCE/PCE+NCE ratio (polychromatic erythrocyte/ polychromatic erythrocyte + normochromatic erythrocyte) in mice bone marrow compared with the non drug-treated irradiated control (p < 0.02-0.00001). The maximum reduction in MnPCEs was observed in mice treated with extract at a dose of 200 mg/kg. Administration of amifostine at dose 100 mg/kg and hawthorn at dose 200 mg/kg reduced the frequency of MnPCE almost 4.8 and 5.7 fold; respectively, after being exposed to 2 Gy of gamma rays, compare with the irradiated control group. Crataegus extract exhibited concentration-dependent activity on 1,1-diphenyl 2-picrylhydrazyl free radical showing that Crataegus contained high amounts of phenolic compounds and the HPLC analysis determined that it contained chlorogenic acid, epicatechin and hyperoside. It appeared that hawthorn extract with antioxidant activity reduced the genotoxicity induced by gamma irradiation in bone marrow cells.     

p53-dependent delayed effects of radiation vary according to time of irradiation of p53 + / ? mice

We previously reported that in p53 ^{+ / −} mice that had been given a whole-body dose of 3 Gy at 8 weeks of age, p53-dependent delayed effects of radiation, as manifested in T-cell receptor (TCR) variant fractions (VF) instability in mouse splenocytes, were biphasic, namely, induction of TCR-VF mutation reappeared at 44 weeks. The manifestation of the delayed effects and the measures of biological markers varied according to the timing of irradiation. We also reported that the decrease in function of the p53 gene was related to the effects of a delayed mutation. In the present study, we investigated the functions and mutations of the p53 gene in old age for p53 ^{+ / −} mice following irradiation at various ages. p53 ^{+ / −} mice were given a whole-body dose of 3 Gy at 8, 28 or 40 weeks of age. There were significant differences for all variables tested at 8 weeks of age. This was similarly the case for mice irradiated at 28 weeks of age, in which there were also significant differences in TCR VF and the percentage of apoptosis. In mice irradiated at 40 weeks of age, there were significant differences for all considered variables except for the p53 allele. We demonstrated that the different patterns of delayed mutation of the p53 gene at 56 weeks of age depended on the age at which mice had undergone 3-Gy whole-body irradiation. Our conclusions are limited to variation in p53-dependent delayed effects according to the time of irradiation.     

CD71荧光抗体和PI染色的微核流式细胞术检测研究

目的探索CD71荧光抗体和PI染色的微核流式细胞术自动化检测方法。方法采用-85℃甲醇固定细胞,CD71荧光抗体和PI染色,以伯氏疟原虫感染小鼠血细胞为微核模式生物调校流式细胞仪,建立微核流式细胞术检测方法,并对秋水仙素(COL)诱导的小鼠外周血含微核网织红细胞进行了检测。结果所建立的方法能明确分辨外周血含与不含微核的网织红细胞和成熟红细胞,COL处理小鼠的外周血含微核网织红细胞率呈明显的剂量依赖性升高,流式细胞术与显微镜检测的外周血含微核网织红细胞率有良好相关性(r=098)。结论微核流式细胞术检测方法能准确地检测小鼠外周血含微核网织红细胞。     

Trp53 activity is repressed in radio-adapted cultured murine limb bud cells

Understanding the effects of of ionizing radiation (IR) at low dose in fetal models is of great importance, because the fetus is considered to be at the most radiosensitive stage of the development and prenatal radiation might influence subsequent development. We previously demonstrated the existence of an adaptive response (AR) in murine fetuses after pre-exposure to low doses of X-rays. Trp53-dependent apoptosis was suggested to be responsible for the teratogenic effects of IR; decreased apoptosis was observed in adapted animals. In this study, in order to investigate the role of Trp53 in AR, we developed a new model of irradiated micromass culture of fetal limb bud cells, which replicated proliferation, differentiation and response to IR in murine embryos. Murine fetuses were exposed to whole-body priming irradiation of 0.3 Gy or 0.5 Gy at embryonic day 11 (E11). Limb bud cells (collected from digital ray areas exhibiting radiation-induced apoptosis) were cultured and exposed to a challenging dose of 4 Gy at E12 equivalent. The levels of Trp53 protein and its phosphorylated form at Ser18 were investigated. Our results suggested that the induction of AR in mouse embryos was correlated with a repression of Trp53 activity.     

p53 dependency of radio-adaptive responses in endogenous spleen colonies and peripheral blood-cell counts in C57BL mice

Radio-adaptive responses at a conditioning X-ray dose of 0.45 Gy and a challenging dose of 5.0 Gy on hematopoietic indices were studied in C57BL mice with p53 (Trp53) wild, heterogenous and knockout allele. The conditioning irradiation, given 2 weeks before the challenging irradiation, induced radio-adaptive responses observed as a recovery of the peripheral blood-cell counts of leukocytes, thrombocytes and erythrocytes on day 14 after challenging irradiation in C57BL mice of the wild-type p53(+/+). The pre-irradiation also increased the endogenous spleen colonies (endo-CFU-S) on day 12 and the spleen weight on day 14. On the contrary, the knockout p53(-/-) mice gave no such radio-adaptive response. The heterogenous p53(+/-) mice gave an intermediate response. The radio-adaptive response in hematopoiesis at a challenge dose of 5.0 Gy seems to be a p53-dependent phenomenon. The possible role of induction in radio-resistance through the reduction of p53-drived apoptosis in hematopoietic stem cells in pre-irradiated mice is discussed.     

Radioprotective effects of citrus extract against gamma-irradiation in mouse bone marrow cells

The radioprotective effects of citrus extract were investigated by using the micronucleus test for anticlastogenic and cell proliferation activity. A single intraperitoneal (i.p.) injection of citrus extract (Citrus aurantium var. amara) at 250, 500, 1000 mg/kg body weight 1 h prior to gamma-ray irradiation (1.5 Gy) reduced the frequencies of micronucleated polychromatic erythrocytes (MnPCE(S)) and normochromatic erythrocytes (MnNCE (S)). All three doses of citrus extract significantly reduced the frequencies of MnPCEs and MnNCEs in mice bone marrow compared to non-drug-treated irradiated control (p < 0.005-0.05). The optimum dose for protection in mouse was 250 mg/kg to protect mice bone marrow 2.2-fold against the side effects of gamma-irradiation with respect to the non-drug-treated irradiated control. The flavonoids were contained in citrus extract, probably to show protective activity, and reduced the clastogenic effect of radiation on mice bone marrow. Therefore fruits and vegetables contain flavonoids to be useful as protective effects under such stress conditions as irradiation.     

Erythrocyte magnesium fluxes in mice with nutritionally and genetically low magnesium status

Summary Low intracellular magnesium (Mg) contents may be observed in case of severe Mg insufficient intake or because of genetic regulation. This work was conducted to investigate the influence of intracellular Mg content on erythrocyte Mg²⁺ influx and efflux in mice with low nutritionally and genetically (MGL and MGH mice) Mg status. C57BL6 mice were fed for 2 wks a diet containing 1000 mg Mg/kg diet Mg (control group), 100 mg Mg/kg diet (Mg–marginal group) or 30 mg Mg/kg diet (Mgdeficient group), while mice with low (MGL) and high (MGH) Mg levels were fed a control diet for 2 wks. The quantification of erythrocyte Mg²⁺ influx and efflux was performed using a stable isotope of Mg. Our results showed that erythrocyte Mg²⁺ influx and efflux were respectively increased and decreased in nutritional Mg deficiency; while in genetically determined Mg status Mg²⁺ fluxes were lower in MGL mice compared to MGH mice. Moreover Mg²⁺ efflux was significantly correlated to Mg level in erythrocytes in all the mice studied (p < 0.001). In conclusion, erythrocyte Mg²⁺ influx and efflux are modulated by low Mg status, namely decreased Mg²⁺ efflux compensate for nutritional Mg deficiency, while the genetic regulation of erythrocyte Mg²⁺ content depends on modification of Mg²⁺ influx.     

Vitamin B-6 metabolic enzymes in blood and placenta of pregnant mice

Plasma pyridoxal 5'-phosphate (PLP) concentrations decrease 50% in pregnant mice and erythrocyte PLP levels increase threefold over nonpregnant levels. These studies were designed to determine whether changes in the enzymes involved in synthesis and degradation of PLP in blood are altered during pregnancy. We measured net synthesis of PLP in erythrocytes and the activity of enzymes involved in the regulation of plasma and erythrocyte PLP concentration: erythrocyte pyridoxal kinase (PLK) and neutral phosphatase, and plasma and tissue alkaline phosphatase (ALP). Net synthesis of PLP and activities of erythrocyte PLK and neutral phosphatase in erythrocytes remained unchanged during pregnancy. We were unable to detect any dephosphorylation of PLP in erythrocytes of pregnant or nonpregnant mice. Mouse erythrocytes were devoid of ALP activity; neutral phosphatase was inactive with PLP and PLP was an uncompetitive inhibitor of the enzyme. Plasma ALP activity decreased 50% in the pregnant mice; therefore, it likely does not participate in the reduction of plasma PLP levels during pregnancy. Placenta had high levels of PLP-phosphatase activity (ALP) and, if it is active as an ectoenzyme in this tissue as it is in others, it may be the most important mediator of plasma PLP levels in pregnancy.     

Ficoll fractionation for the separation of parasitized erythrocytes from malaria infected blood.

Separation and concentration of parasitized erythrocytes from infected blood was achieved by centrifugation of a sample placed in a layer on top of a cushion of a Ficoll solution with a critical density. Pure suspensions of parasitized erythrocytes were obtained from Plasmodium berghei infected rodent blood, whereas results with P. vivax infected monkey (Aotus trivirgatus) blood were partially successful. Titration experiments revealed that the parasitized erythrocytes obtained by Ficoll fractionation were infective.