Metabolism of glutathione in tumour cells as evidenced by 1H MRS.

1H MRS signals of glutathione and of free glutamate were examined in samples from cultured tumour cells, namely MCF-7 from mammary carcinoma and TG98 from malignant glioma, with the aim of relating signal intensities to aspects of GSH metabolism. Spectra of cells harvested at different cell densities suggest that GSH and glu signal intensities are related to cell density and proliferation and their ratio is dependent on the activity of the gamma-glutamyl cysteine synthetase. The hypothesis is confirmed by experiments performed on cells treated with buthionine sulfoximine that inhibits the enzyme activity.

1H MRS signals from glutathione may act as predictive markers of apoptosis in irradiated tumour cells.

Inhibition of apoptosis in tumour cells may depend on intracellular reduced glutathione (GSH) level. In this work, GSH levels were studied by (1)H MRS in MCF-7 and HeLa cells, characterised by a different radiosensitivity. Annexin-V test showed that the fraction of apoptotic HeLa cells after irradiation is much higher than in control, although MCF-7 cells did not show a significant apoptosis. MRS signals from GSH (G) show lower intensity in HeLa with respect to MCF-7 cells; the opposite is true for free glutamic acid [glu (g)]. After irradiation, the G/g ratio decreases in MCF-7, although remaining approximately constant in HeLa cells. Buthionine sulfoximine (BSO) treated MCF-7 cells show an increase in the percentage of apoptotic cells; in parallel, G/g ratio behaves as in HeLa. This study indicates that GSH level may act as predictive marker of apoptosis by irradiation.

Radiation effects in cultured tumour cells examined by 1H MRS: mobile lipids modulation and proliferative arrest.

Much attention has been devoted in the past to monitor changes of mobile lipid (ML) (1)H MRS signals in spectra of tumour cells. The purpose of this work is to exploit ML signals to provide information on cell metabolism after irradiation, comparing tumour cells characterised by different radiosensitivity and relating MRS findings to changes in cell proliferation and delays in cell cycle phases. Irradiated HeLa cells present less intense ML signals with respect to controls. The opposite is true for MCF-7 cells. A G(2) arrest is observed for both cell lines after irradiation. In HeLa cells, G(1) decreases and S phase is maintained; a sub G(1) peak is also visible. In MCF-7 cells, G(1) is decreased and S phase is strongly reduced, while no sub G(1) is present. The observed changes in ML are tentatively associated to cell cycle regulation of phospholipid synthesis. Mathematical modelling of ML variations is in progress.

Membranes modification of differentiating proerythroblasts. Variation of 1,6-diphenyl-1,3,5-hexatriene lifetime distributions by multifrequency phase and modulation fluorimetry.

The fluorescence emission of 1,6-diphenyl-1,3,5-hexatriene (DPH) in K562 cell membranes has been studied using multifrequency phase and modulation fluorimetry. The DPH decay data collected at various modulation frequencies were analysed by assuming either a model of discrete exponential components or a model of continuous lifetime distribution. The fits showed smaller values of the reduced chi square using the model of continuous lifetime distribution. The K562 cell membranes dynamics were investigated during the cell differentiation along the erythroid pathway. By using the continuous lifetime distribution method for the analysis of the DPH decay, marked variations were observed during the four initial days of the erythroid differentiation. Namely, the width of the DPH lifetime distribution increased by a factor of about two, while the center value of the distribution remained constant. By using the discrete exponential components model for the analysis of the DPH decay no variations were observed during the K562 differentiation.

Fluorescence studies using synchrotron radiation on normal and differentiated cells labeled with parinaric acids.

Changes in membrane properties during the differentiation process in K562 cells have been investigated. A decrease of lectin-induced agglutination has been detected. The agglutination assay revealed to be an early and sensitive test to monitor the induced differentiation of the K562 cells. Naturally occurring fluorescent fatty acids (cis- and trans-parinaric acids) and the recently developed multifrequency phase and modulation technique were used to study cell membrane properties. Changes in fluorescence lifetime and polarization are clearly associated with cell differentiation, suggesting the involvement of the cellular plasma membrane in the differentiation process.

Cholesterol protects the phospholipid bilayer from oxidative damage.

The measurement of fluorescence lifetime distribution of 1,6-diphenyl-1,3,5-hexatriene is used for the detection of oxidative damage produced in phospholipid membranes by ionizing radiation. The recently developed method is based on the linear relationship between the width of the probe lifetime distribution and the logarithm of the dose. The molecular origin of the damage resides in the production of hydroperoxide residues at the level of acyl chains double bonds. A chemiluminescence assay was used to quantitate the amount of produced hydroperoxides. Consequences of the produced damages include an increased disorder in the upper portion of the bilayer, accompanied by the penetration of water molecules. In the presence of the physiological concentration of cholesterol in phopholipid bilayers, the amount of hydroperoxides produced by ionizing radiation is dramatically reduced. The packing effect of cholesterol in phopholipid bilayers is well recognized, as well as its influence on the reduction of water concentration in the bilayer. The dramatic reduction of hydroperoxides concentration observed when irradiation is performed in the presence of cholesterol probably originates from a steric hindrance to the radical chain reaction through the unsaturated lipids due to the presence of cholesterol.

In K562 and HL60 cells membrane ageing during cell growth is associated with changes in cholesterol concentration.

In a cell culture model of aging we have previously shown that there is an age-related decrease in the lipid dynamics of the proerythropoetic K562 cell membranes, as determined by the generalized polarization (GP) of the phase-sensitive lipid probe 2-dimethylamino-6-lauroylnaphthalene (Laurdan) (T. Parasassi, M. Di Stefano, G. Ravagnan, O. Sapora and E. Gratton. Exp. Cell Res., 202 (1992) 432-439). In the present study we also extended our observations to the lymphoblastoid HL60 cell line. In both K562 and HL60 cells during the four days after the last cell culture medium renewal the GP Laurdan value increased in a linear fashion indicating a time-dependent decrease in lipid dynamics. The initial membrane physical properties were almost completely restored upon renewal of the cell culture medium. We measured lipid composition, including individual and total phospholipids, free and esterified cholesterol at the first ('young') and at the fourth ('aged') day after culture medium renewal. We found that the decreased membrane lipid 'fluidity' at the fourth day of cell growth was associated with a 40% increase in cholesterol concentration in both cell lines. This increase in cholesterol concentration was reversible 24 h following the culture medium change. We conclude that in K562 and HL60 cells the 'age-related' decrease in membrane lipid dynamics is mediated by an 'age-related' increase in cell cholesterol content.

Enhancement of DNA damage in mammalian cells upon bioreduction of the nitroimidazole-aziridines RSU-1069 and RSU-1131.

The induction of DNA double-(dsb) and single-(ssb) strand breaks by RSU-1069, RSU-1131 and misonidazole in V79 mammalian cells has been investigated using sedimentation in isokinetic sucrose gradients after incubation for various times (1-3 hr) at 310 K under both hypoxic and aerobic conditions. Double strand breaks are produced by RSU-1069 and RSU-1131 predominantly under hypoxic conditions. Comparison of the cellular DNA damage induced by these agents leads to the following facts: (1) the yield of ssb induced by these agents is substantially increased under hypoxia, (2) RSU-1069 and RSU-1131 are much more effective than misonidazole, on a concentration basis, at causing strand breakage both under hypoxic and aerobic conditions; and (3) RSU-1069 is more efficient on a concentration basis than RSU-1131 at inducing both ssb and dsb under both conditions. From these findings and molecular studies it is suggested that these 2-nitroimidazole aziridines act as monofunctional alkylating agents under aerobic conditions, a factor that governs their aerobic cytotoxicity. Under hypoxic conditions, it is suggested that the induction of dsb and crosslinks by these agents (bifunctional character) may play a major role in determining the ability of such agents to act as hypoxia-selective cytotoxins.

The repair of DNA damage induced in V79 mammalian cells by the nitroimidazole-aziridine, RSU-1069. Implications for radiosensitization.

The induction and repair of single (ssb) and double (dsb) strand breaks in DNA under aerobic or hypoxic conditions have been determined using sucrose sedimentation techniques following incubation of V79 mammalian cells with RSU-1069 or misonidazole, representative of a conventional 2-nitroimidazole radiosensitizer, for 1-1.5 hr at either 293 or 277 degrees K and subsequent irradiation at 277 degrees K. In all cases, the dose dependences for the induction of strand breaks are linear and consistent with an enhancement in the yield of DNA damage induced by the 2-nitroimidazoles under hypoxic conditions. With RSU-1069 at 293 degrees K, the dose dependence of ssb is displaced reflecting DNA damage induced during pre-incubation. From these dependences, it is evident that the enhanced radiosensitization by RSU-1069 may not be accounted for in terms of accumulation of the agent at DNA. From the repair studies, DNA breaks induced by RSU-1069 in the absence of radiation have been shown to persist for at least 3 hr. With a combination of RSU-1069 and radiation under hypoxic conditions, the repair timescale of the induced breaks is significantly longer and an increase in the residual yields of both ssb and dsb (at 2-3 hr) was observed when compared with the observation in the presence of misonidazole or oxygen. From these studies, it is inferred that the enhanced radiosensitization of RSU-1069 at 293 degrees K is a consequence of the formation of non-repairable DNA damage together with a modification of the repairability of the radiation-induced DNA breaks.

Induction of mutations in V79-4 mammalian cells under hypoxic and aerobic conditions by the cytotoxic 2-nitroimidazole-aziridines, RSU-1069 and RSU-1131. The influence of cellular glutathione.

Incubation of the 2-nitroimidazole-aziridine, RSU-1069 [1-(2-nitro-1-imidazolyl)-3-(1-aziridinyl)-2-propanol], and its monomethylaziridine analogue, RSU-1131 [1-(2-nitro-1-imidazolyl)-3-(1-(2-methylaziridinyl))-2-propanol], with V79-4 mammalian cells for 2 hr under aerobic or hypoxic conditions induces mutations as measured at the hypoxanthine phosphoribosyl transferase locus. The ability of these agents to induce mutations is increased by a factor of 12-14 under hypoxic conditions. The increased cytotoxicity of these agents under hypoxic conditions was confirmed following a 2 hr incubation period. Decreasing the glutathione (GSH) content of the cells with buthionine-(S,R)-sulphoximine to < 1% of the control generally results in an increase in the cytotoxicity and mutagenicity of these agents under both aerobic and hypoxic conditions. Since these agents do not modify the cellular GSH levels, it is inferred that the thiols partially detoxify through removal of a reactive metabolite of the agents, under hypoxic conditions, or removal of known DNA adducts, and not through their interaction with the agents themselves. Under aerobic conditions, the formation of mutations is consistent with the established monofunctional action of these agents whereas under hypoxic conditions the bifunctional action predominates for mutation induction, based upon the large differential aerobic:hypoxic effect. From a comparison of the number of mutations per lethal event, the effect of thiol depletion is more pronounced for cytotoxicity than for mutation induction by these agents. In summary, these agents are considered to be weak mutagens towards V79-4 cells under aerobic conditions when compared with other DNA alkylating agents, although they are more potent under anoxic conditions.

Membrane oxidative damage induced by ionizing radiation detected by diphenylhexatriene fluorescence lifetime distributions.

The sensitivity of the fluorescence lifetime of 1,6-diphenyl- 1,3,5-hexatriene (DPH) to the dielectric constant of its environment has been used to detect oxidative damage to phospholipid membranes induced by ionizing radiation. The DPH fluorescence decay in phospholipid vesicles is described well by a continuous distribution of lifetime values, reflecting the various DPH depths in the bilayer and related to the gradient of the dielectric constant. Ionizing radiation oxidizes unsaturated acyl residues of phospholipids, altering the dielectric constant across the bilayer, sharpening the distribution of DPH lifetimes and increasing the centre of the distribution. Ionizing radiation doses between 22 and 110 Gy were used, and were effective only in the presence of oxygen. A model based on the formation of packing defects in the bilayer describes the phenomenon.

Human cell membrane oxidative damage induced by single and fractionated doses of ionizing radiation: a fluorescence spectroscopy study.

PURPOSE: To investigate the production and repair of lipid oxidative damage in two human cell lines exposed to acute and fractionated dose of ionizing radiation. Radiation dose was in the range from 0.1 to 44 Gy. MATERIALS AND METHODS: K562 and HL60 human cell lines have been used, 24 and 96 h after seeding. Membrane lipid oxidative damage has been detected by the measurement of the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH), its polarization value and the conjugated dienes concentration. The modification of DPH decay has been previously reported to be directly related to the lipid hydroperoxide concentration. RESULTS: A modification of the DPH decay has been observed as a linear function of the logarithm of the radiation dose and only when the irradiation was performed in the presence of oxygen. The amount of the damage is related to the time after the cell medium change. By exposing the cells to fractionated radiation doses for several days (10 cGy day(-1)), the oxidative damage has been found to be cumulative. After a single acute dose, evidence of repair of the lipid oxidative damage was not obtained. CONCLUSIONS: Following a previously developed method, the membrane damage was attributed to the production of hydroperoxide residues in the lipid acyl chains with the consequence of water penetration into the external portion of the bilayer, from the aqueous environment to the position of hydroperoxides. This damage is not repaired. The results obtained by measuring the DPH fluorescence decay have been compared with those obtained using other current optical and biochemical methods. None of these techniques could detect membrane oxidative damage at doses < 10 Gy. Finally, the different sensitivity of 'young' and 'old' cells to the oxidative damage can be related to different cholesterol concentrations.

Evidence for an increase in water concentration in bilayers after oxidative damage of phospholipids induced by ionizing radiation.

The two membrane fluorescent probes 2-dimethyl-amino-6-lauroyl-naphthalene (Laurdan) and 2-dimethylamino-6-propionyl-naphthalene (Prodan) have been used to study the molecular basis of the damage induced in phospholipid membranes by ionizing radiation. Laurdan and Prodan display a spectral sensitivity to the polarity of their environment, showing a red shift of both excitation and emission spectra with increase of the polarity of their environment. Owing to their chemical differences, the two probes are anchored in the membrane with different strengths. In aqueous environments Laurdan is not fluorescent while Prodan shows appreciable fluorescence. Laurdan and Prodan show an opposite response to oxidative damage produced in phospholipid bilayers by ionizing radiation. The results support the model recently developed of water penetration in the bilayer as a consequence of oxidative damage.

Alterations in erythrocyte membrane lipids induced by low doses of ionizing radiation as revealed by 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime.

Damage in membrane lipids induced by low doses of ionizing radiation in the presence of oxygen has been detected in rabbit erythrocyte ghosts labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH). Multifrequency phase and modulation fluorometry was used to measure DPH fluorescence lifetime. This technique is particularly suited for the observation of heterogeneous fluorescence decays. DPH decay in erythrocyte membranes is described by a two-component continuous distribution of lifetimes. The value of the distribution width of the long-lived component is found to be affected by radiation-induced membrane lipid damage at doses as low as 0.5 Gy, well within the dose range used to measure cell survival. The width of the DPH lifetime distribution decreases when the ghosts are irradiated in the presence of oxygen. Such a decrease is a linear function of the logarithm of the dose. After a dose of 110 Gy and above, the fractional intensity of the short-lived component of the DPH decay increases linearly, indicating severe membrane damage. Experiments performed in the absence of oxygen do not show any change in the fluorescence parameters up to a dose of 550 Gy. The molecular identification of the produced damage has not been accomplished, but the necessity of oxygen to observe the damage suggests that hydroperoxides and lipids crosslinks are produced.

Relationships between cell killing, mutation induction and DNA damage in X-irradiated V79 cells: the influence of oxygen and DMSO.

The relationships between cell killing, mutation induction and DNA double (dsb) and single (ssb) strand breaks have been studied in V79 cells irradiated with X-rays under oxic and anoxic conditions in the presence and in the absence of dimethylsulphoxide (DMSO). Curvilinear relationships were found between all pairs of endpoints, except for dsb versus ssb. Statistical analysis of experimental data has shown that in the absence of DMSO there is evidence of good correlations between cell killing, mutation induction and dsb in oxic and anoxic conditions. However, when DMSO was present, no significant correlation was found. In the presence of oxygen DMSO always exerts a protective effect while in anoxia it is generally much less protective and induces a strong sensitization with respect to mutation induction. Possibly DMSO acts not only as a radical scavenger but also as an agent inducing chromatin relaxation and/or under anoxia, forming highly mutagenic short-term radicals. The present data suggest that lethal and mutational events are at least partially independent and not proportional to the initial number of DNA breaks. This may imply that either other kinds of lesions are involved in cell lethality and mutability, or dose-dependent repair mechanisms of dsb have to be considered.

RBE-LET relationship for the survival of V79 cells irradiated with low energy protons.

The survival of V79 Chinese hamster cells irradiated with proton beams with energies of 0.73, 0.84, 1.16, 1.70 and 3.36 MeV, corresponding to LET values, evaluated at the cell midplane, of 34.5, 30.4, 23.9, 17.8 and 10.6 keV/micron respectively, have been studied in the dose range 0.5-6.0 Gy. As a reference, the survival curve obtained with 200 kV X-rays was used. The initial shoulder, typical of survival curves obtained with sparsely ionizing radiation, decreases as the LET increases and completely disappears at 23.9 keV/micron. This value corresponds to the maximum of the RBE, expressed as the initial slope ratio. In the energy range we have considered, the RBEs for protons are higher than those reported for other ions of comparable LET and the RBE-LET relationship results shifted to lower LET values. Our data seem to indicate that the RBE-LET curve depends on the type of radiation and this could imply that LET is not a good reference for the dose-effectiveness relationship.

DNA double-strand breaks induced by low energy protons in V79 cells.

The initial production of DNA double-strand breaks (dsb) was determined in V79 Chinese hamster cells irradiated with proton beams of 3.24, 1.50 and 0.88 MeV, corresponding to values of unrestricted LET evaluated at the cell midplane of 10.9, 20.0 and 30.5 keV/micron, respectively. X-rays were used for comparison. Dsb were measured with the low speed sedimentation technique in neutral sucrose gradients. The initial yield of dsb rose linearly with the dose and did not significantly depend on the proton LET, in contrast with the results obtained in previous studies for cell inactivation and mutation induction. Also, no significant differences for dsb induction were found between protons and X-rays. Two possible explanations, not necessarily mutually exclusive, are proposed: (1) dsb are not the only lesions involved in cellular effects; and (2) the initial number of dsb is not the only important parameter since a fundamental role is played by the degree of clustering, i.e. the association of dsb with other dsb or other types of damage.

RBE-LET relationships for cell inactivation and mutation induced by low energy protons in V79 cells: further results at the LNL facility.

PURPOSE: RBE-LET relationships for cell inactivation and hprt mutation in V79 cells have been studied with mono-energetic low-energy proton beams at the radiobiological facility of the INFN-Laboratori Nazionali di Legnaro (LNL), Padova, Italy. MATERIALS AND METHODS: V79 cells were irradiated in mono-layer on mylar coated stainless steel petri dishes, in air. Inactivation data were obtained at 7.7, 34.6 and 37.8 keV/microm and hprt mutation was studied at 7 7 and 37.8 keV/microm. Additional data were also collected for both the end points with the proton LET already considered in our previous publications, namely 11.0, 20.0 and 30.5 keV/microm. RESULTS: A maximum in the RBE-LET relationship for cell inactivation was found at around 31 keV/microm, while the RBE for mutation induction increased continuously with LET. CONCLUSIONS: The proton RBE-LET relationship for cell inactivation is shifted to lower LET values compared with that for heavier ions. For mutation induction, protons of LET equal to 7.7keV/microm gave an RBE value comparable with that obtained by helium ions of about 20 keV/microm. Mutagenicity and lethality caused by protons at low doses in the LET range 7.7-31 keV/microm were proportional, while the data at 37.8 keV/microm suggest that this may not hold at higher LET values.

Mutation induction and RBE-LET relationship of low-energy protons in V79 cells.

The mutation induction at the HGPRT locus has been studied in V79-753B Chinese hamster cells irradiated with proton beams with energies of 3.36, 1.70 and 1.16 MeV, corresponding to average LET values of 10.6, 17.8 and 23.9 keV/microns, respectively. The mutation curve obtained with 200 kV X-rays was used for comparison. The mutation frequency induced by all the proton beams is considerably higher than that induced at the same dose by X-rays and it is linearly related to the dose. Moreover, the proton effectiveness increases with the LET. The RBEs (evaluated as the initial slope ratios) are 5.0 +/- 0.8, 5.4 +/- 0.8 and 7.7 +/- 1.2 for protons with average LETs of 10.6, 17.8 and 23.9 keV/microns, respectively. These values are higher than those reported in the literature for other ions of comparable LET. This finding parallels what we have already found for cell inactivation (for which RBEs of 3.0, 4.6 and 7.3 were obtained at the same LETs), and indicates that for mutation induction, also, the RBE-LET relationship may depend on the type of radiation.

DNA fragmentation in V79 cells irradiated with light ions as measured by pulsed-field gel electrophoresis. I. Experimental results.

PURPOSE: To compare the results on DNA fragmentation induced in Chinese hamster V79 cells by various doses of gamma-rays and low-energy protons and helium-4 ions. MATERIALS AND METHODS: V79 cells were irradiated as monolayers with monoenergetic protons and helium-4 ions; gamma-rays were used as the reference radiation. DNA double-strand breaks were evaluated by calibrated pulsed-field gel electrophoresis using conditions covering the range 5.7 Mbp-23.1 kbp. RESULTS: The fragment-counting method gave double-strand breaks yields and the relative biological effectiveness higher than those obtained by the fraction of activity released method. The frequency distribution of fragments showed that protons and helium ions induced more fragments below the Mbp region than did gamma-rays at the same dose. The distributions for both the irradiated and non-irradiated samples clearly appeared to be non-random. CONCLUSION: Differences were observed in the yield and spatial correlation, at a molecular size scale characteristic of loop dimensions, of the double-strand breaks induced by gamma-rays and by light ions. These effects may have a role in the observed different cell response to these radiations.