Bleomycin sulfate-induced micronuclei in human,rat, and mouse peripheral blood lymphocytes

The sensitivity to micronucleus (MN) induction of human, mouse, and rat peripheral blood lymphocytes (PBLs) exposed to bleomycin sulfate (BLM) in vitro was compared in cytochalasin B-induced binucleated (BN) cells. For the PBLs of each species, either 0, 5, 10, 20, 40, 60, 80, or 160 μg/ml BLM was added to 5 ml aliquots of whole blood for 4 hr at 37°C in a 5% CO₂ atmosphere. Leukocytes were isolated on a density gradient and cultured in the presence of phytohemagglutinin to stimulate blastogenesis, and cytochalasin B was added to each culture at 21 hr postinitiation to prevent cytokinesis. A total of 4,000 BNs/concentration/species was analyzed for MN in two independent experiments. In addition, multiple-MN-BNs were quantitated, and the nucleation index was determined. Significant increases both in total MN-BNs and multiple-MN-BNs were observed at all concentrations in all species. All three species concentration-response curves gave good fits (r² values from 0.87 to 0.95) to either a linear or a square root model (y = mx + b or y = m[x]^0.5 + b, respectively; where y = the percentage of MN-BN, m is the slope, and b is the y-intercept). The MN induction in the human and rat PBLs was not statistically different, but both were significantly less sensitive than the response shown by the BLM-exposed mouse PBLs. This difference in MN susceptibility was observed only at BLM test concentrations ≧ 20 μg/ml. The nucleation index was significantly decreased in all species at either 80 or 160 μg/ml. © 1995 Wiley-Liss, Inc.     

Interspecies cytogenetic comparisons: studies with X-radiation and bleomycin sulfate.

A series of in vitro experiments were conducted to determine if there are innate differences in the sensitivity of peripheral blood lymphocytes (PBLs) from different mammalian species to clastogens. Mouse, rat, and human whole blood samples were exposed to either 0, 0.38, 0.75, 1.5, or 3.0 Gy x-radiation or 0, 5, 10, 20, 40, or 80 micrograms/ml bleomycin for 4 hr. Bromodeoxyuridine-containing cultures were initiated and the PBLs stimulated to divide with phytohemagglutinin. All cultures were harvested following a 3-hr colcemid treatment. Slides were made and differentially stained, and first-division metaphases were scored for chromosome aberrations. In the x-radiation studies human PBLs were significantly more sensitive than mouse PBLs which were in turn more sensitive than rat PBLs as measured by either the total percent aberrant cells or the number of dicentrics. Data from all three species could be fitted to a linear-quadratic model. Results with bleomycin suggest that the mouse and human PBLs are equally sensitive to the clastogenic effects of bleomycin. Both appeared to be more sensitive than the rat PBLs, but the variation between experiments was such that the results among species were not significantly different. These results indicate that there may be inherent differences in sensitivity among PBLs of mammalian species; however, more studies are needed to determine if the differences presented here hold for other agents.     

Micronucleus induction in cytokinesis-blocked mouse bone marrow cells in vitro following in vivo exposure to x-irradiation and cyclophosphamide

A cytokinesis-block micronucleus (MN) method for the simultaneous but separate measurement of chromosome damage in erythroid and myeloid bone marrow cells is described. MN induction in cytokinesis-blocked mouse bone marrow cells in vitro following in vivo exposure to x-ray or cyclophosphamide (CP) was investigated. Immediately after whole body irradiation with acute doses of either 0, 1, 2 or 4 Gy x-rays, or 2 hr after treatment with either 0, 12.5, 25, or 50 mg CP/kg body weight, bone marrow cells were collected and then cultured in medium supplemented with 3.0 μ/ml cytochalasin B for 24 hr. The binucleated cells were scored in erythroid, myeloid, lymphoid and other cells. The myeloid/erythroid (M/E) ratio was decreased by x-irradiation or CP treatment in a dose-dependent manner. The dividing index (DI; binucleated cells/binucleated + mononucleated cells; %) was decreased in both eryth-roid and myeloid cells in the same manner. Dose-dependent increases in MN frequency were observed following x-irradiation in both erythroid and myeloid cells. A similar dose-dependent MN induction was observed with CP. The MN frequency in myeloid cells was much greater than in erythroid cells (about 4-fold following 4 Gy exposure, and more than 10-fold after 50 mg/kg CP). Lymphoid and other cells were not suitable for scoring DI and MN frequency because of insufficient numbers of binucleated cells. These results suggest that micronuclei can be identified in both myeloid and erythroid cells and that myeloid cells are more susceptible to x-ray or CP-induced chromosomal damage than erythroid cells as expressed by MN induction. © 1994 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.     

Dietary folate deficiency enhances induction of micronuclei by arsenic in mice

Folate deficiency increases background levels of DNA damage and can enhance the genotoxicity of chemical agents. Arsenic, a known human carcinogen present in drinking water supplies around the world, induces chromosomal and DNA damage. The effect of dietary folate deficiency on arsenic genotoxicity was evaluated using a mouse peripheral blood micronucleus (MN) assay. In duplicate experiments, male C57Bl/6J mice were fed folate-deficient or folate-sufficient diets for 7 weeks. During week 7, mice on each diet were given four consecutive daily doses of sodium arsenite (0, 2.5, 5, or 10 mg/kg) via oral gavage. Over the course of the study the folate-deficient diet produced an approximate 60% depletion of red blood cell folate. Folate deficiency by itself was associated with small but significant increases in MN in normochromatic erythrocytes (NCEs) and polychromatic erythrocytes (PCEs). Arsenic exposure was associated with significant increases in MN-PCEs in both folate-deficient and folate-sufficient mice. MN-PCE frequencies at the 10 mg/kg dose of arsenic were increased 4.5-fold over vehicle control in folate-deficient mice and 2.1-fold over control in folate-sufficient mice. At the 5 and 10 mg/kg doses of arsenic, MN-PCE levels were significantly higher (1.3-fold and 2.4-fold, respectively) in folate-deficient mice compared to folate-sufficient mice. Very few MN from either control or treated animals in either experiment exhibited kinetochore immunostaining, suggesting that the MN were derived from chromosome breakage rather than from whole chromosome loss. These results indicate that folate deficiency enhances arsenic-induced clastogenesis at doses of 5 mg/kg and higher.     

Studies on the genotoxicity of molybdenum salts in human cells in vitro and in mice in vivo

Molybdenum is an essential element in plants and animals as a cofactor for enzymes. Molybdenum trioxide is used in metallurgical processes, in cosmetics as a pigment, and in contact lens solution, yet limited information is available on molybdenum genotoxicity. In the present study the micronucleus (MN) assay in human lymphocytes and mouse bone marrow and the dominant lethal assay in mice were used to assess the genotoxic effects of molybdenum salts in vitro and in vivo. Two salts of molybdenum were tested in whole blood cultures. Ammonium molybdate was more potent than sodium molybdate in causing a dose-dependent decrease in viability and replicative index and an increase in MN formation in binucleated lymphocytes (P < 0.001). A dose–response in both kinetochore-positive MN (caused by chromosome lagging) and kinetochore-negative MN (associated with chromosome breakage) was observed. Based on the results of a toxicity study of sodium molybdate, two doses, 200 and 400 mg/kg, were assessed in the bone marrow MN assay in mice (two i.p. injections 24 and 48 hr prior to euthanasia). A modest but statistically significant increase in MN frequency in polychromatic erythrocytes was observed (P < 0.05). The same treatment protocol was used to analyze dominant lethality. A dose-dependent increase in postimplantation loss represented mostly by early resorptions was observed the first week after treatment (P = 0.003). These preliminary data suggest that sodium molybdate induces dominant lethality at the postmeiotic stage of spermatogenesis. Overall, molybdenum salts produced moderately positive results both in vitro in human cells and in vivo in mice. Environ. Mol. Mutagen. 32:251–259, 1998 © 1998 Wiley-Liss, Inc.     

Micronuclei induction by dichlorvos in the mouse skin.

Micronucleus (MN) induction in cultured keratinocytes was investigated following skin painting of HRA/Skh mice with the pesticide, dichlorvos. Whole skin and partially-purified epidermal cells from 5-6 week old male animals were cultured for 4 days in vitro after single topical applications of various concentrations of dichlorvos in vivo. Appropriate doses, allowing optimum survival of keratinocytes, were selected following an initial range-finding experiment. To evaluate MN induction in dividing cells, the cytokinesis-block method was employed. Results showed statistically significant MN at all dose levels in partially-purified epidermal cells and a positive trend with respect to dose from 51 to 1033 nmol dichlorvos. A significant increase in MN was also detectable in cultured cells from whole skin, dissociated within as little as 1 h after application of dichlorvos. Although a number of technical difficulties are associated with the skin micronucleus method, it has been used successfully in this laboratory to detect several skin carcinogens of both high and low potency. Since dichlorvos is rapidly absorbed through the skin, and can induce MN in skin cells of treated mice, this compound may therefore be considered to pose a contact hazard for exposed humans.     

Flow cytometric scoring of micronucleated reticulocytes as a possible high‐throughput radiation biodosimeter

Micronucleated reticulocyte (MN‐RET) scoring by flow cytometry (FCM) has been used in assessment of the clastogenic effects of chemicals. However, its dose–response to acute whole body irradiation (WBI) at moderate dose rates remains to be established. We show that FCM scoring of MN‐RET in peripheral blood from male ICR mice exposed to WBI X‐ray doses of 0.5–5 Gy at a dose rate of 0.488 Gy/min exhibits a linear dose–response relationship 24, 48, and 72 hr following WBI. Parallel microscopic counting of micronucleated polychromatic erythrocytes (MN‐PCE) in bone marrow smears from the same animals showed similar linear dose–response patterns at the same time points. Indeed, MN‐RET and MN‐PCE were highly correlated at all doses and time points. In view of the speed and accuracy of this method, in addition to the small blood sample size needed for the assay, the flow cytometric protocol for MN‐RET scoring may provide a minimally‐invasive, high throughput radiation biodosimeter. Environ. Mol. Mutagen., 2010. © 2009 Wiley‐Liss, Inc.     

Induction of 6-thioguanine-resistant lymphocytes in Fischer 344 rats following in vivo exposure to N-ethyl-N-nitrosourea and cyclophosphamide

We have developed a limiting dilution clonal assay for determining the frequency of 6-thioguanine-resistant (TGr) lymphocytes produced in rats by in vivo exposure to genotoxic agents. Spleen lymphocytes were isolated from female Fischer 344 rats and were cultured with 1 microgram/ml of phytohemagglutinin (PHA) for 40 hr. Northern blot analysis revealed that this procedure resulted in increased hprt and beta-actin mRNA synthesis. Conditions for optimum cloning were established by culturing four PHA-primed lymphocytes/well in 96-well round-bottom microtiter plates containing a medium supplemented with interleukin-2. These cultures also contained autologous and/or TK6 feeder cells inactivated with different doses of irradiation. Lymphocyte cloning efficiencies (CEs) were highest in plates containing both irradiated TK6 cells (5 x 10(3) cells/well; 90 Gy) and irradiated autologous feeder cells (5 x 10(4) cells/well; 50 Gy). CE did not depend on the number of primed lymphocytes/well when four or fewer target cells/well were cloned. To measure the effects of chemical mutagens on the frequency of TGr lymphocytes, rats were given a single i.p. injection of 0-150 mg/kg of N-ethyl-N-nitrosourea (ENU), a direct-acting alkylating agent, or 0-50 mg/kg of cyclophosphamide (CP), an indirect acting alkylating agent. Lymphocytes were isolated, primed, and cloned at 4 weeks after CP treatment and at 1, 2, 4 and 6 weeks after ENU treatment. CE in these cultures ranged from 12% to 27%. Cultures were also established for measuring CE in the presence of 6-thioguanine (TG) and these contained 5 x 10(3) irradiated TK6 cells and 5 x 10(4) primed rat lymphocytes/well. The frequency of TGr lymphocytes was calculated by correcting the CE in the presence of TG with the CE measured in its absence. ENU exposure produced a higher frequency of TGr lymphocytes than CP, but both chemicals produced a dose-dependent increase in TGr cells. In addition, the frequency of ENU-induced TGr lymphocytes increased with time after treatment. The TGr cells are presumed to be hprt mutants, but further analysis at the DNA level is required to establish this. The dose-dependent responses obtained with both ENU and CP treatments suggest that rat lymphocytes are sensitive to direct- and indirect-acting alkylating agents administered in vivo and that the rat lymphocyte assay is a useful complement to the in vivo/in vitro mouse assay for determining the mutagenicity of environmental toxicants.     

放射性胸腺损伤小鼠模型的建立

背景：胸腺对于维持机体免疫功能起着重要的作用,在放射性损伤发生时的损伤和修复机制有待于进一步研究,但目前缺乏相应的动物模型。 目的：单纯照射小鼠胸腺以构建放射性胸腺损伤模型。 方法：160只雌性BALB/C小鼠随机分为4组,每组40只,对照组予以假照射;6 Gy照射组给予⁶⁰Co γ射线6 Gy胸腺单次照射,9 Gy照射组给予胸腺单次照射9 Gy,12 Gy照射组给予胸腺单次照射12 Gy。观察各组小鼠照射后1-7 d体质量及进食水量变化,照射后1,7,14,21,28 d检测各组小鼠血象、胸腺指数及胸腺病理变化,并于照射后7 d检测小鼠食管、肺、气管病理组织学变化,照射后14 d检测小鼠外周血T细胞亚群和胸腺T细胞亚群表达。 结果与结论：与对照组相比,照射组小鼠各项指标均发生变化,但6 Gy照射组由于损伤相对较轻,在照后1周内就开始修复;12 Gy照射组出现了放射性食管、气管损伤,而9 Gy组即保留了放射性损伤的特点,也避免了其他脏器损伤带来的干扰,能反映胸腺放射性损伤后的改变。以9 Gy ⁶⁰Co γ射线单次纵膈照射可成功制作小鼠放射性胸腺损伤模型。     

Studies in vivo of cobra factor and murine C3.

The effect of the isolated C3-cleaving factor (CoF) of cobra venom on murine C3 in vivo and in vitro was studied. Optimal quantities of 100-200 units (0.5 minus 1.0 mg) of CoF per kg administered to mice by intraperitoneal injection in divided doses caused plasma C3 levels to fall to less than 5 per cent of normal from 1 to at least 4 days afterwards. Passive anti-CoF serum promptly abrogated the in vivo plasma C3 depletion, and under optimal conditions C3 levels reached 50 per cent of normal after approximately 15 hours. Injection of as little as 20 mug per mouse of CoF in saline induced a precipitating anti-CoF antibody response which prevented subsequent depletion of plasma C3 by CoF. The in vivo half-life of 125I-labelled CoF in normal mice estimated by whole body elimination and clearance from the blood was 24 hours. The presence in vivo of antibodies to CoF caused rapid clearance from the blood and elimination of 125I-labelled CoF, and also localization of some CoF in the spleen, liver and kidneys.     

Assessment of the ability of propoxur,methomyl, and aldicarb,three carbamate insecticides,to induce micronuclei in vitro in cultured Chinese hamster ovary cells and in vivo in BALB/c mice

Three carbamate insecticides (propoxur, methomyl, and aldicarb) were evaluated for their ability to induce micronuclei (MN) in vitro using cultured Chinese hamster ovary (CHO) cells, and in vivo in mouse bone marrow erythrocytes. In vitro, all three insecticides induced a significant increase in micronucleated binucleate cells, which was generally both dose and sample time dependent. The in vivo studies involved treating male BALB/c mice by different routes, either once or on 3 consecutive days, followed by multiple or single sampling. Treatment by intraperitoneal injection or oral gavage induced a significant increase in micronucleated reticulocytes (MNRETs) in peripheral blood. For all three chemicals, the MN response depended on sample time and the number of treatments, while for aldicarb, the response depended also on the route of exposure. These positive results demonstrate that propoxur, methomyl, and aldicarb are capable of inducing structural and/or numerical chromosomal aberrations in mammalian cells either in vitro or in vivo. Furthermore, based on the results obtained, an optimal in vivo MN protocol for carbamate insecticides is a single treatment followed by blood sampling at 24 and 48 hr after treatment. Environ. Mol. Mutagen. 29:386–393, 1997 © 1997 Wiley-Liss, Inc.     

Evaluation of the mutagenicity of the anti-inflammatory drug salicylazosulfapyridine (SASP)

Salicylazosulfapyridine, commonly known as sulfasalazine or SASP, is an anti-inflammatory drug that is widely used in the treatment of diseases such as ulcerative colitis and Crohn's disease. Increases in sister chromatid exchanges (SCE) and micronuclei (MN) frequencies have been reported in lymphocytes of patients maintained on SASP therapy for up to 21 months. We have tested SASP for its ability to induce chromosome aberrations (ABS) and SCE in cultured Chinese hamster ovary (CHO) cells, ABS in mouse bone marrow cells, and MN in erythrocytes from both bone marrow and peripheral blood of mice. In vitro assays for ABS and SCE were negative. In vivo, SASP administered by single gavage at doses up to 1000 mg/kg did not increase ABS in bone marrow cells of male B6C3F1 mice; however, increases in MN were observed in the peripheral blood erythrocytes of male and female B6C3F1 mice administered 675, 1350 or 2700 mg/kg SASP by gavage for 90 days. Weak but significant dose-related increases in MN were also observed in the bone marrow cells of male B6C3F1 mice administered 500, 1000 and 2000 mg/kg SASP for 3 days. These positive findings in mice support the role of SASP in the induction of MN and SCE in humans, and suggest the need for further evaluation of possible adverse human health effects associated with SASP therapy.     

蝎毒多肽对辐射损伤小鼠骨髓造血干细胞及祖细胞的作用

目的 探讨不同蝎毒多肽(scorpion venom peptide,SVP)组分对辐射后机体造血干细胞及祖细胞恢复的作用.方法 6.0 Gy X射线一次性全身照射,制作辐射损伤小鼠模型.内源性脾结节法观察照射后第10天脾集落形成单位(CFU-S)的变化.用甲基纤维素半固体培养基培养骨髓混合集落生成单位(CFU-Mix),观察体内外给药方法及照射后不同时间对CFU-Mix生成的影响.结果 (1)体内实验:SVPⅣ组分处理后的CFU-S数明显高于照射对照组(P＜0.05);SVPV组分CFU-S数量与照射对照组差异无统计学意义.照射后各SVP组CFU-Mix的数量均高于照射对照组,差异有统计学意义(P＜0.05).(2)体外实验:与照射对照组相比,体外分别单独加入SVPⅣ、Ⅴ组分以及细胞因子(IL-6和SCF)均能够促进CFU-Mix的增殖;而SVPⅣ、Ⅴ组分分别与细胞因子联合应用对CFU-Mix生成的促进作用更为明显,其中Ⅳ组分效果更强,与照射对照组相比差异均有统计学意义(P＜0.05).结论 SVP具有保护辐射损伤小鼠造血干细胞及祖细胞,加速其增殖能力恢复的作用.     

Effect of Biostim (RU 41.740) on natural killer cell generation from bone marrow precursors

We have evaluated the possible effect of RU 41.740 (Biostim), a mixture of two glycoproteins extracted from K. pneumoniae, on the in vitro interleukin-2 (IL-2)-induced generation of NK cells from bone marrow (BM) precursors and on the in vivo reconstitution of splenic NK activity in lethally irradiated (9 Gy) and BM reconstituted mice. Our results show that RU 41.740 is able to augment the generation of NK cells when added (1-0.01 micrograms/ml) to normal or 5-fluorouracil-resistant BM, cultured in the presence of recombinant IL-2. Also, in vivo treatment of lethally irradiated mice, transplanted with syngeneic BM cells, with RU 41.740 (1-0.1 mg/kg i.v.) from day 0 through day 4 after BM graft, resulted in a significant augmentation of NK activity reconstitution.     

Catalytic inhibitors of topoisomerase II are DNA-damaging agents: induction of chromosomal damage by merbarone and ICRF-187

Merbarone is a catalytic inhibitor of topoisomerase II (topo II) that has been proposed to act primarily by blocking topo II-mediated DNA cleavage without stabilizing DNA-topo II-cleavable complexes. In this study merbarone was used as a model compound to investigate the genotoxic effects of catalytic inhibitors of topo II. The clastogenic properties of merbarone were evaluated using in vitro and in vivo micronucleus (MN) assays combined with CREST staining. For the in vitro MN assay, ICRF-187, a different type of catalytic inhibitor, and etoposide, a topo II poison, were used for comparison. Treatment of TK6 cells with all three of these drugs resulted in highly significant dose-related increases in kinetochore-lacking MN and, to a lesser extent, kinetochore-containing MN. In addition, a good correlation between p53 accumulation and MN formation was seen in the drug-treated cells. A mouse MN assay was performed to confirm that similar DNA-damaging effects would occur in vivo. Bone marrow smears from merbarone-treated B6C3F1 mice showed a dose-related increase in micronucleated polychromatic erythrocytes with a mean of 26 MN per 1000 cells being seen at the 60 mg/kg dose. Almost all MN lacked a kinetochore signal, indicating that merbarone was predominantly clastogenic under these conditions in vivo. The present study clearly shows that merbarone is genotoxic both in vitro and in vivo, and demonstrates the inaccuracy of earlier statements that merbarone and other catalytic inhibitors block the enzymatic activity of topo II without damaging DNA.     

Micronucleus induction in mouse and rat fetuses treated transplacentally during histogenesis with mitomycin C and 7,12-dimethylbenz(a)anthracene

During histogenesis, mouse and rat fetuses were treated transplacentally with mitomycin C (MMC) and 7,12-dimethylbenz(a)anthracene (DMBA). The micronucleus (MN)-inducing effects of MMC were analysed in mouse fetal blood and liver; the effects of DMBA were analysed in mouse fetal and rat fetal blood and in maternal bone marrow of both species. Both test substances were clearly clastogenic during the period of development in which the embryos were analysed--i.e., MMC from gestational day 14 until day 18 in mice and DMBA on days 14 and 17 in mice and days 16 and 19 in rats. In mouse fetal liver and blood the MMC-induced MN frequencies did not vary significantly during the whole period. MMC was more effective in fetal blood than in fetal liver. DMBA-induced MN frequencies in maternal bone marrow were slightly higher in rats than in mice. Compared to maternal bone marrow, fetal MN frequencies were about four to five times higher in rats but less than two times higher in mice. Thus, rat fetuses were far more susceptible to the clastogenic action of DMBA than mouse fetuses. These results are discussed with respect to fetal development and maternal/fetal metabolism.     

Resistance and Susceptibility to Cyclosporin A as CD3− 4− 8− Human Thymocytes Differentiate In Vitro

Human T-cell development appears to be relatively resistant to cyclosporin A (CsA). Children exposed to CsA mw/eroaspart of kidney transplant maintenance have few abnormalities. The objective of the study described here was to analyse the effects of CsA on the development in vitro of human multinegative (MN) (CD3-4-8-) thymocytes as a model system for thymic progenitor development in vitro. MN thymocytes, prepared by depletion methods, differentiated in vitro to acquire CD3 and undergo transitions in CD45 isoform expression analogous to those postulated to occur in vivo. In this work MN thymocytes were cultured with IL-2 and on thymic epithelial cells (TEC) with or without lL-2, either in ihe presence or absence of CsA. For many thymocyte preparations, differentiation in the presence of CsA resulted in almost complete inhibition of the acquisition of CD3and ofthe low Mr isofomi CD45R0. Expression of CD45RA and of total CD45 were reduced but not eliminated and the density of CD29 was unaffected. For others, neither CD3 nor CD45 expression was affected., but selective inhibition of TCR5 expression occurred. At all doses of CsA (0.1–100 μg/ml), MN thymocytes continued to cycle indicating a CsA-resistant generative compartment. Treatment of peripheral blood T cells with CsA had no effect on surface expression of CD3 or CD45 isoforms but did reduce the amount of de novo-synthesized CD45R0 mRNA. Culture of MN thymocytes on TEC rendered them virtually resistant to the negative effects of CsA. CD3 acquisition was unhindered and total CD45 remained high, but the transition from CD45RA to CD45R0 appeared to be delayed. In the absence of TEC, expression of both TCRδ and of TCRδ was inhibited, but on TEC, TCRδ was actually up-regulated in some conditions. The effects of CsA on human thymocyte development appeared to be modulated by the physiological state of the donor and the growth conditions to which the ceils were subjected. Conditions which most closely approximated those manifest in vivo rendered thymocytes most resistant to the negative effects of CsA. The amount of CsA required to affect differentiation in vitro was significantly higher than could be attained in vivo suggesting that the immunomodulatory effects of CsA in the maintenance of organ transplants may from an as yet uncharacterized mechanism     

中药抗突变实验研究

本文从1984年起分别以C57纯种小鼠的骨髓细胞和人外周血淋巴细胞为实验材料，用公认的诱变剂环磷酰胺(CPP)诱发的姐妹染色单体互换(SCE)，微核(MN)升高作指标，对大量中草药进行实验，已筛选出部分补气药或相关物以及各种人参的有效成分或单体具有抗突变作用。     

Ionizing radiation accelerates the development of atherosclerotic lesions in ApoE-/- mice and predisposes to an inflammatory plaque phenotype prone to hemorrhage

After radiotherapy treatment, there is an increased incidence of localized atherosclerosis in patients with Hodgkin's disease, breast cancer, and head and neck cancer. Here, we established a mouse model to study the development and progression of radiation-induced atherosclerosis and to compare the phenotype of these lesions with age-related atherosclerosis. Atherosclerosis-prone ApoE-/- mice fed a regular chow diet received single radiation doses of 14 Gy or sham treatments (0 Gy) to the neck, including both carotid arteries. At 22, 28, and 34 weeks after irradiation, blood samples were taken, and the arterial tree was removed for histological examination. Cholesterol levels in irradiated mice were not significantly different from age-matched controls, and markers of systemic inflammation (soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, and C-reactive protein) were not elevated. The lesions in irradiated arteries were macrophage rich, with a remarkable influx of inflammatory cells, predominantly granulocytes. Intraplaque hemorrhage and erythrocyte-containing macrophages were seen only in lesions of irradiated arteries. Based on these data, we propose that irradiation accelerates the development of macrophage-rich, inflammatory atherosclerotic lesions prone to intraplaque hemorrhage.