Membrane oxidative damage induced by ionizing radiation detected by fluorescence polarization

Effects of ionizing radiation on biological membranes include alterations in membrane proteins, peroxidation of unsaturated lipids accompanied by perturbations of the lipid bilayer polarity. We have measured radiation-induced membrane modifications using two fluorescent lipophilic membrane probes (TMA-DPH and DPH) by the technique of fluorescence polarization on two different cell lines (Chinese hamster ovary CHO-K1 and lymphoblastic RPMI 1788 cell lines). γ-Irradiation was performed using a ⁶⁰Co source with dose rates of 0.1 and 1 Gy/min for final doses of 4 and 8 Gy. Irradiation induced a decrease of fluorescence intensity and anisotropy of DPH and TMA-DPH in both cell lines, which was dose-dependent but varied inversely with the dose rate. Moreover, the fluorescence anisotropy measured in lymphoblastic cells using TMA-DPH was found to decrease as early as 1 h after irradiation, and remained significantly lower 24 h after irradiation. This study indicates that some alterations of membrane fluidity are observed after low irradiation doses and for some time thereafter. The changes in membrane fluidity might reflect oxidative damage, thus confirming a radiation-induced fluidization of biological membranes. The use of membrane fluidity changes as a potential biological indicator of radiation injury is discussed. Received: 14 May 1996 / Accepted in revised form: 30 September 1996     

Fluidity of intact erythrocyte membranes. Correction for fluorescence energy transfer from diphenylhexatriene to hemoglobin

Membranes of intact erythrocytes were labeled by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) using an improved labeling procedure described previously (Plásek, J. and Jarolím, P. (1987) Gen. Physiol. Biophys. 6, 425-437). The relationship between the steady-state DPH fluorescence anisotropy r and the mean corpuscular hemoglobin concentration (MCHC) was studied. Fluorescence anisotropy increased with increasing MCHC. A linear dependence of r = 0.0026 (MCHC) + 0.113 was obtained which enabled us to measure the fluidity of intact red cell membranes. Without this correction for fluorescence quenching by hemoglobin, incorrect conclusions about membrane fluidity could be made. This fact is demonstrated in a group of pyruvate kinase deficient patients compared with a group of healthy blood donors.     

Alterations in erythrocyte membrane fluidity in children with trisomy 21: a fluorescence study.

Membrane fluidity of erythrocytes obtained from 15 children with trisomy 21 and 20 healthy controls were studied by measuring steady-state fluorescence anisotropy and fluorescence lifetime of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated in hemoglobin-free erythrocyte membranes. Our results demonstrate a significant decrease in DPH fluorescence anisotropy and a significant increase in TMA-DPH fluorescence anistropy in erythrocytes from subjects with trisomy 21. No significant differences between the two groups were observed in the fluorescence lifetime of DPH and TMA-DPH. These data suggest an increase in membrane fluidity in the interior part of the membrane and a decrease in fluidity at the lipid-water interface region. This could be in part attributed to an increased oxidative damage in trisomy 21.     

Alterations in Membrane Lipid Dynamics of Leukemic Cells Undergoing Growth Arrest and Differentiation: Dependency on the Inducing Agent

The effect of various differentiation inducers on membrane cell dynamics was studied using HL-60 and K562 leukemic cell lines. Membrane lipid dynamics was measured by the steady-state fluorescence polarization (P) method utilizing either 1,6-diphenyl-1,3,5-hexatriene (DPH) or the trimethyl ammonium derivative of DPH (TMA-DPH), which ascertains anchorage of the label to the membrane–water–lipid interface. Decrease in membrane microfluidity was observed in HL-60 cells undergoing differentiation into macrophages by 1,25-dihydroxyvitamin D₃and by K562 cells induced to differentiate by DMSO. Sodium butyrate caused an increase in membrane fluidity in K562 cells undergoing differentiation into erythroid-like cells while in HL-60 cells a dual effect was observed. At 0.4 mM concentration, in which the cells were induced to differentiate along the monocyte pathway, a decrease in membrane fluidity was observed, while at 1 mM concentration an increase in membrane fluidity occurred. Interferon-γ (IFN-γ) induced an increase in membrane fluidity in both cell lines. Using HL-60 cells fluorescently labeled by TMA-DPH, similar results indicating fluidization of the membrane following IFN-γ treatment were obtained. Advanced fluorescence lifetime measurements, evaluated either by phase modulation spectrofluorometry or by single photon correlation fluorometry confirmed that the decrease in fluorescence polarization by IFN-γ resulted from membrane fluidization and not from elongation of the probe's excited state lifetime. It is suggested that the inducer mode of action, and not the differentiation route, determine the outcome of changes in membrane microviscosity.     

Changes in plasma membrane fluidity of corn (Zea mays L.) roots after Brij 58 treatment

Summary The detergent Brij 58 has been introduced to reverse plasma membrane (PM) vesicles from the right-side-out to the inside-out form. The aim of the present work was to investigate the effect of Brij 58 on the formation of an ATP-dependent proton gradient and on the fluidity of the lipid phase of PM vesicles. PMs of corn (Zea mays L.) roots were isolated by phase-partitioning. The fluidity of PMs was estimated by measurement of fluorescence polarization with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The PMs of corn roots were relatively rigid. The hydrophobic part of the lipid bilayer was more fluid than the hydrophilic part. After intercalation of Brij 58 into the lipid bilayer the membrane fluidity changed in a concentration-dependent manner. Treatment with the detergent Brij 58 increased the degree of fluorescence polarization for TMA-DPH, while it decreased it for DPH. This effect was saturated at a detergent-to-protein ratio of 1 4 for both fluorescence probes. Although the biophysical characteristics of the membrane were changed after Brij 58 treatment, the formation of ATP-dependent proton gradients could still be measured with those vesicles. The generation of an ATP-dependent proton gradient with Brij 58-treated PM vesicles suggests that the detergent treatment indeed turned the originally right-side-out vesicles to sealed inside-out vesicles. The limits of the effect caused by Brij 58 in the context of PM enzyme activities are discussed.Abbreviations Brij 58 polyoxyethylene 20 cetyl ether - DPH 1,6-diphenyl-1,3,5-hexatriene - HCF III hexacyanoferrate (III) - ISO inside-out - PM plasma membrane - RSO right-side-out - TMA-DPH 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene     

Activation of human lymphocytes by concanavalin A or purified protein derivative results in no alteration of fluorescence polarization of lipid probes although the electrophoretic mobility of the cells is changed

Upon stimulation with either concanavalin A or the tuberculin antigen, purified protein derivative, human peripheral blood lymphocytes, purified on Ficoll-Hypaque, did not exhibit a concomitant lipid fluidity alteration as measured by fluorescence polarization (P) of the lipid probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). This result was independent of the incubation period, ranging from 10 min to 72 h. However, a general reduction in polarization value, from P = 0.287 (maintained for up to 2 h of incubation) to P = 0.225 after 20 h was observed for both experimental and control samples. Moreover, fluorescence polarization studies of the nonpenetrating modified DPH cationic lipid probe, 1-[4′-trimethylaminophenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), also failed to show any change in lipid fluidity subsequent to a 1–3 h incubation of lymphocytes with concanavalin A. Cell electrophoretic mobility, however, was altered (mean cell mobility increased by 10–15%) in a fast response to stimulation and was observed within several hours of in vitro application of concanavalin A and purified protein derivative. This initial response disappeared with further incubation at 37°C (>3 h) and was followed by a decline of cellular mobility of the concanavalin A-exposed cells after 48 and 72 h of incubation. The unstimulated control cells did not change in mobility as a function of incubation time. The slow decline in mean cell mobility of the experimental cells is believed to be associated with blastogenesis. It is concluded that neither blastogenic transformation nor short term membrane alterations associated with human lymphocyte activation lead to lipid fluidity changes as measured in steady state by the fluorescence polarization of both DPH and TMA-DPH.     

Comparative study on fluorescent probes distributed in human erythrocytes and platelets

Important cellular functions, such as rheological properties of cells are presumably related to the membrane lipid fluidity which may be approached by the use of fluorescence polarization method. However, biological membranes represent very heterogeneous media and the knowledge of the fluidity of membrane compartments requires the use of different probes. Two fluorescent probes, DPH and its cationic derivative, TMA-DPH, have been employed to probe the lipid fluidity of human platelets and red cell membranes. The results show that the informations given by DPH and TMA-DPH can present important differences, suggesting that DPH and TMA-DPH are localized in different regions of cell membranes. In an attempt to investigate relations between lipid fluidity and rheological properties of red cells, the behavior of probes was studied in a "Couette" viscometer with a device for studying the emissive properties of probes when red cell membranes are under shear conditions.     

Membrane fluidity measured by fluorescence polarization using an EPICS V cell sorter

We have characterized the measurement of fluorescence polarization on single cells using an EPICS V cell sorter. A critical analysis is made of the balancing and calibration of the system. The system is highly linear for polarization measurements. Cellular membranes were labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) to measure membrane fluidity. Fluorescence polarization histograms had coefficients of variation as low as 7%. Cells labeled with DPH after 24 hr incubation in medium lacking serum showed a significantly higher fluorescence polarization than cells in medium containing serum. The fluorescence polarization measured at 15 degrees C was 0.311 compared to 0.270 at 25 degrees C for cells labeled with DPH, verifying that temperature affects the membrane fluidity as measured by flow cytometry.     

Static and dynamic components of renal cortical brush border and basolateral membrane fluidity: Role of cholesterol

Summary Static polarization and differential polarized phase fluorimetry studies on rat renal cortical brush border (BBM) and basolateral membranes (BLM) were undertaken to determine the membrane components responsible for differences in BBM and BLM fluidity, whether these differences were due to the order or dynamic components of membrane fluidity and if a fluidity gradient existed within the bilayer. Surface membrane proteins rigidified both BBM and BLM fluidity. Neutral lipid extraction, on the other hand, caused a larger decrease in BBM than BLM fluorescence polarization (0.104vs. 0.60,P<0.01) using diphenyl hexatriene (DPH). Cholesterol addition to phospholipid fractions restored membrane fluidity to total lipid values in both BBM and BLM phospholipids. The response to cholesterol in the BBM was biphasic, while the BLM response was linear. Lateral mobility, quantitated using dipyrenylpropane, was similar in both BBM and BLM fractions at 35°C. BBM and BLM differed primarily in the order component of membrane fluidity as DPH-limiting anisotropy (r ) (0.212vs. 0.154,P<0.01) differed markedly between the two membrane fractions. The two membrane components also differed with respect to 2 and 12-anthroyloxy stearate (2-AS, 12-AS) probes, indicating a difference in the dynamic component of membrane fluidity may also be present. DPH and 12-As probes were also used to quantitate inner core membrane fluidity and showed the BBM was less fluid than the BLM for intact membranes, total lipid extracts and phospholipids. Results obtained using the surface membrane probes trimethylammonium-DPH (TMA-DPH) and 2-AS suggested a fluidity gradient existed in both BBM and BLM bilayers with the inner core being more fluid in both membranes. These data indicate cholesterol is in large part responsible for fluidity differences between BBM and BLM and that these membranes, while clearly differing in the order component of membrane fluidity, may also difer in the dynamic component as well.     

DPH标记细胞膜的动力学与膜脂流动性的荧光偏振校正测量

用稳态荧光技术测得经过校正的荧光成分,由此算出用DPH标记的细胞膜的偏振度。方法是作荧光偏振值在随时间变化的曲线,将其外推至零标记时间求出该时间的荧光偏振值。用此法测定了艾氏腹水癌细胞的膜流动性。结果表明流动性比用整个细胞测得之值小,说明膜脂的有序程度和包装密度比胞浆中的脂大。实验结果和用三房空模型分析所得的理论值符合较好,提示荧光探剂的标记过程主要受分子扩散所控制。     

呼吸链底物和抑制剂对线粒体内膜流动性的影响

用DPH和ANS标记大鼠肝线粒体内膜,以稳态荧光偏振法,研究了呼吸链底物和抑制剂对内膜流动性的影响。1.苹果酸+谷氨酸、琥珀酸分别为底物,均能引起内膜流动性增加。2.琥珀酸对含心磷脂的脂质体的膜流动性无影响。3.在鱼藤酮存在的条件下,苹果酸+谷氨酸对内膜流动性的增加作用消失,但琥珀酸的作用仍然存在。有氰化钾时则琥珀酸的作用消失。4.不论外加底物存在与否,鱼藤酮使内膜的流动性下降,而氰化钾则使之增加。抗霉素A亦可使内膜的流动性增加。上述结果表明:线粒体内膜流动性与其功能密切相关。电子沿呼吸链传递使线粒体内膜流动性增加,这种变化可能与呼吸链成分的氧化还原态有关。     

Glucocorticoid-mediated alterations in fluidity of rabbit cardiac muscle microvessel endothelial cell membranes: influences on eicosanoid release

Experiments were conducted to determine effects of the synthetic glucocorticoid, dexamethasone, on the lipid fluidity of cultured rabbit cardiac muscle microvessel endothelial cells and the possible role(s) for altered fluidity in the steroid inhibition of cellular eicosanoid production. Following a sixteen hour exposure to 10(-7) M dexamethasone, membranes prepared from treated cells exhibited a decreased fluidity compared to their control counterparts, as assessed by steady-state fluorescence polarization techniques using 1,6-diphenyl-1,3,5-hexatriene (DPH). Examination of the effects of temperature on the anisotropy values of DPH using Arrhenius plots revealed consistent differences in the steroid treated cells over the entire temperature range (40-5 degrees C). These dexamethasone-dependent fluidity changes were associated with increases in the cholesterol/phospholipid ratio of membrane lipids. Restoration of membrane fluidity to control values with the fluidizing agent, 2-(2-methoxyethoxy)ethyl-8-(cis- 2-n-octylcyclopropyl)octanoate (A2C), partially reversed dexamethasone induced inhibition of A23187-stimulated eicosanoid release. These observations suggest that at least part of dexamethasone's inhibitory actions on eicosanoid generation in microvessel endothelial cells are mediated by alterations in membrane composition and fluidity.     

Effect of amyloid β-peptide on the fluidity of phosphatidylcholine membranes: Uses and limitations of diphenylhexatriene fluorescence anisotropy

There is accumulating evidence that peptide-induced perturbations in the order and dynamics of cellular membranes may play a role in the neurotoxicity of amyloid β-peptide (Aβ). Several studies have reported that Aβ decreases fluidity of membranes based on an Aβ-induced increase in the fluorescence anisotropy of diphenylhexatriene (DPH). However, the effect of Aβ on the membrane fluidity is still a subject of controversy, because other studies that employed pyrene as a fluorescent probe have shown that Aβ has the opposite effect. To reveal the reason for this discrepancy, we have examined the effect of Aβ on the fluidity of phosphatidylcholine membranes using spectroscopic methods. The fluorescence anisotropy of DPH is dramatically increased on addition of Aβ to DPH-containing phosphatidylcholine membranes. However, Aβ does not affect the Raman spectrum of the membrane, which is sensitive to the packing order of the hydrocarbon chains of lipids. We have also found that circular dichroism (CD) bands of DPH appear during incubation of DPH-containing membranes with Aβ, whereas DPH is an achiral molecule. The observed CD bands of DPH are induced by a chiral environment of Aβ but not by that of the lipids, because positive CD bands appear regardless of the d/l-chirality of phosphatidylcholine. The findings obtained from CD measurements provide evidence that DPH molecules translocate from the membrane to Aβ. The peptide-mediated extraction of DPH from the membrane may cause changes in the fluorescence anisotropy of DPH, even though Aβ does not affect the fluidity of membranes.     

Fluorescence anisotropy analysis of the mechanism of action of mesenterocin 52A: speculations on antimicrobial mechanism

Mesenterocin 52A (Mes 52A) is a class IIa bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, active against Listeria sp. The interaction of Mes 52A with bacterial membranes of two sensitive Listeria strains has been investigated. The Microbial Adhesion to Solvents test used to study the physico-chemical properties of the surface of the two strains indicated that both surfaces were rather hydrophilic and bipolar. The degree of insertion of Mes 52A in phospholipid bilayer was studied by fluorescence anisotropy measurements using two probes, 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and DPH, located at different positions in the membrane. TMA-DPH reflects the fluidity at the membrane surface and DPH of the heart. With Listeria ivanovii CIP 12510, Mes 52A induced an increase only in the TMA-DPH fluorescence anisotropy, indicating that this bacteriocin affects the membrane surface without penetration into the hydrophobic core of the membrane. No significant K⁺ efflux was measured, whereas the ΔΨ component of the membrane potential was greatly affected. With Listeria innocua CIP 12511, Mes 52A caused an increase in the fluorescence of TMA-DPH and DPH, indicating that this peptide inserts deeply in the cytoplasmic membrane of this sensitive strain. This insertion led to K⁺ efflux, without perturbation of ΔpH and a weak modification of ΔΨ, and is consistent with pore formation. These data indicate that Mes 52A interacts at different positions of the membrane, with or without pore formation, suggesting two different mechanisms of action for Mes 52A depending on the target strain.     

巴氏碳球C60光激发对红细胞膜流动性的影响

巴氏碳球Ｃ_（６０）光激发对红细胞膜流动性的影响黄文栋,钱凯先,唐海琼（浙江大学生物科学与技术系,杭州３１００２７）李文铸（渐江大学物理系,杭州３１００２７）关键词Ｃ_（６０）;光激发;红细胞膜;荧光偏振;膜流动性Ｃ６０是Ｋｒｏｔｏ等人［１］于１９８５...     

Chloroplast thylakoid membrane fluidity and its sensitivity to temperature

In order to investigate membrane fluidity, the hydrophobic probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), has been incorporated into intact isolated thylakoids and separated granal and stromal lamellae obtained from the chloroplasts of Pisum sativum. The steady-state polarization of DPH fluorescence was measured as a function of temperature and indicated that at physiological values the thylakoid membrane is a relatively fluid system with the stromal lamellae being less viscous than the lamellae of the grana. According to the DPH technique, neither region of the membrane, however, showed a sharp phase transition of its bulk lipids from the liquid-crystalline to the gel state for the temperature range -20° to 50° C. Comparison of intact thylakoids isolated from plants grown at cold (4°/7°C) and warm (14°/17° C) temperatures indicate that there is an adaptation mechanism operating which seems to maintain an optimal membrane viscosity necessary for growth. Using a modified Perrin equation the optimal average viscosity for the thylakoid membrane of the chill-resistant variety used in the study (Feltham First) is estimated to be about 1.8 poise.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - Hepes N-(2-hydroxyethyl)-1-piperazineethanesulphonic acid     

The effect of Pycnogenol on the erythrocyte membrane fluidity

In the present study, the in vitro effect of polyphenol rich plant extract, flavonoid--Pycnogenol (Pyc), on erythrocyte membrane fluidity was studied. Membrane fluidity was determined using 1-[4-trimethyl-aminophenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), 1,6-diphenyl-1,3,5-hexatriene (DPH) and 12-(9-anthroyloxy) stearic acid (12-AS) fluorescence anisotropy. After Pyc action (50 microg/ml to 300 microg/ml), we observed decreases in the anisotropy values of TMA-DPH and DPH in a dose-dependent manner compared with the untreated erythrocyte membranes. Pyc significantly increased the membrane fluidity predominantly at the membrane surface. Further, we observed the protective effect of Pyc against lipid peroxidation, TBARP generation and oxidative hemolysis induced by H2O2. Pyc can reduce the lipid peroxidation and oxidative hemolysis either by quenching free radicals or by chelating metal ions, or by both. The exact mechanism(s) of the positive effect of Pyc is not known. We assume that Pyc efficacy to modify effectively some membrane dependent processes is related not only to the chemical action of Pyc but also to its ability to interact directly with cell membranes and/or penetrate the membrane thus inducing modification of the lipid bilayer and lipid-protein interactions.     

Membrane fluidity of Toxoplasma gondii: a fluorescence polarization study

Toxoplasma gondii membrane fluidity was investigated by fluorescence polarization. We used 1,6-diphenyl 1,3,5-hexatriene (DPH) as a fluorescent hydrophobic probe. Fluorescence anisotropy (r) and degree of order (s) showed high fluidity properties. Chemical analysis was performed on this parasite. We found a low cholesterol/phospholipid ratio, many unsaturated fatty acids chains, and high phosphatidylcholine and low sphingomyelin amounts. These results were in good agreement with the observed high fluidity. This may be related to the great adaptability of Toxoplasma gondii in infesting a wide variety of host cells.     

抗生素对莱氏衣原体膜流动性和Mg~(2+)-ATPase活性的影响

本文以莱氏衣原体AIH089为材料,用DPH荧光偏振等技术研究红霉素和土霉素对莱氏衣原体膜流动性和Mg~(2+)-ATPase活性的影响,并用聚丙烯酰胺梯度凝胶电泳技术进一步分析膜蛋白的组成,发现红霉素和土霉素能使莱氏衣原体膜的流动性显著增加,使Mg~(2+)-ATPase活性显著降低。红霉素和土霉素对莱氏衣原体膜流动性和膜上Mg~(2+)-ATPase活性的影响与它们的抑菌能力有很好的相关性。     

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy (r) by DPH probe where lower (r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid ((r): 0.263+/-0.001, 0.279+/-0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) ((r): 0.206+/-0.001) (P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes ((r): 0.231+/-0.001, 0.243+/-0.002, respectively) than those incubated with LP-HDL and nat-HDL ((r): 0.223+/-0.001) (P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy (r) and free cholesterol efflux from these cells (-0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.