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.

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.

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.

Optical images of dose distributions in Gel-Fricke: dosimetric performances of the gel.

The purpose of this work was to examine the dosimetric performances of the radiochromic Fricke-Agarose-Xylenol Orange gel by optical measurements in order to perform dose reconstructions, in view of a future development for 3-D maps. Optical images and dose-response curves of the gel were obtained by a CCD-based device, originally designed for reading radiochromic films, that was modified to meet the optical properties of the dosemeter. With a resolution of 0.18 x 0.18 mm the optimum range of doses in which per cent uncertainty is lower than 2% was 3-10 Gy. The minimum detectable dose, estimated as the absorbed dose corresponding to 3 SD above background, was 0.1 Gy. With a resolution of 1.98 x 1.98 mm the optimum range of doses in which per cent uncertainty is lower than 2% was 0.3-10 Gy. The minimum detectable dose, estimated as the absorbed dose corresponding to 3 SD above background, was 0.015 Gy. The comparison with alanine dosemeters in the dose range 7-10 Gy showed agreement within a few per cent and the same agreement was observed for the comparison with TLD in the range 1-3 Gy.

Ion diffusion modelling of Fricke-agarose dosemeter gels.

In Fricke-agarose gels, an accurate determination of the spatial dose distribution is hindered by the diffusion of ferric ions. In this work, a model was developed to describe the diffusion process within gel samples of finite length and, thus, permit the reconstruction of the initial spatial distribution of the ferric ions. The temporal evolution of the ion concentration as a function of the initial concentration is derived by solving Fick's second law of diffusion in two dimensions with boundary reflections. The model was applied to magnetic resonance imaging data acquired at high spatial resolution (0.3 mm) and was found to describe accurately the observed diffusion effects.

Optical imaging of dose distributions in Fricke gels.

Ferrous-sulphate infused gels, or 'Fricke gels', encounter great interest in the field of radiation dosimetry, due to their potential for 3D radiation dose mapping. Typically, magnetic resonance (MR) relaxation rates are determined in these systems in order to derive the absorbed dose. However, when large concentration gradients are present, diffusion effects before and during the MR imaging may not be negligible. In these cases, optical techniques may represent a viable alternative. This paper describes research aimed at measuring 3D dose distributions in a Fricke-xylenol orange gel by measuring optical density with a CCD camera. This method is inexpensive and fast. A series of early experiments is described, in which optical density profiles were measured with a commercial microdensitometer for film dosimetry. The light box of the device was modified to work at 567 nm, close to the maximum absorbance of the ferric ion-xylenol orange complex. Under these conditions, the gel shows linearity with dose and high sensitivity.

Changes in cell volume and internal sodium concentration in HeLa cells during exponential growth and following lonidamine treatment.

Cell volumes decreased in HeLa cells as a function of time after seeding during exponential growth. Cell volume distributions revealed the presence of two cell populations in all stages of growth. When cells approached confluence, the ratio of the two populations abruptly shifted towards that characterised by the smallest volume. Percentages of G1-, S- and G2 + M-phase cells were also measured and it was found that G1 frequency increased as a function of cell density during exponential growth. Intracellular sodium concentration, [Na]i was monitored by 23Na NMR in the presence of 5 mM dysprosium (III) tripolyphosphate. [Na]i increased from 22.8 to 59.0 mM in cells from the second to the seventh day after seeding. Treatment with lonidamine, an antitumoral drug that it is known to slow down cell growth by affecting aerobic glycolysis, produced a complete block of cell progression after a few days of treatment. The progression of cell volume distributions towards smaller volumes and the increase in internal sodium concentration as a function of time after seeding were also affected by the drug. These phenomena were related to the existence of a subpopulation of mitotically inactive G1-phase cells during exponential growth, pointing out that a density-dependent cellular mechanism regulates the cell cycling in HeLa cells.

Characterization of vesicles, containing an acylated oligopeptide, released by human colon adenocarcinoma cells. NMR and biochemical studies.

RNA-containing vesicles, recovered from the supernatant of high-density cell samples of human colon carcinoma, produce a high-resolution 1H NMR spectrum of lipids characterized by isotropic tumbling; these vesicles contain large amounts of triglycerides and cholesterol esters. Both findings have strict analogies to what is displayed by the proteolipid complexes isolated from the sera of tumor-bearing patients [(1985) Proc. Natl. Acad. Sci. USA 82, 3455-3459; (1986) FEBS Lett. 203, 164-168]. Lipid analysis and enzymatic tests indicate that these vesicles are selected micromaps of plasma membranes, analogous to those that can be recovered from culture media in which tumor cells are grown [(1985) Dev. Biol. 3, 33-57]. Peculiar lipids, an acylated oligopeptide and a modified phospholipid, are also present in the vesicles.

Interaction of HIV-1 with susceptible lymphoblastoid cells. 1H NMR studies.

Different strains of HIV susceptible lymphoblastoid cells have been infected by HIV-1 and examined by means of 1H NMR spectroscopy at different times after infection, taking advantage of the presence of high resolution lipid signals from the plasma membrane of tumor cells. A transient decrease in intensity of fatty acid signals, originated by changes in membrane structure, has been observed early after viral infection. Marked alterations in membrane-dependent steps of phospholipid synthesis can also be inferred by the observed transient depression in peaks from choline-based metabolites. Spectral modifications deriving from changes in lipid metabolism are also produced both in infected cells a few days after infection and in permanently infected cells. 1H NMR can, therefore, monitor structural and metabolic effects induced by HIV infection.

Metabolic and structural effects of HIV infection in human peripheral blood mononuclear cells can be monitored with 1H NMR spectroscopy.

Infection of human peripheral blood lymphocytes by human immunodeficiency virus type 1 (HIV-1) was investigated by means of 1H nuclear magnetic resonance spectroscopy, taking advantage of the presence of signals from fluid lipid domains in the membrane of stimulated lymphocytes. A transient decrease of the lipid methylene signal intensity was observed at the time of HIV internalization, monitoring a general rearrangement of membrane structure associated with virus entry. A similar effect was also observed a few days after infection, when HIV particles are released by infected cells as demonstrated by high reverse transcriptase activity in cell supernatant. Signals arising from choline-based metabolites were also affected by HIV infection, indicating a possible slowing down of phospholipid synthesis.

RNA-lipid complexes released from the plasma membrane of human colon carcinoma cells.

Cultured cells from human colon adenocarcinoma spontaneously release structures which display an intense 31P NMR signal from RNA and mobile phospholipids. Furthermore, the DPH probe in the cell supernatant shows an intense fluorescence, thus indicating its insertion in lipid vesicles. The total membranes, prepared from the same cells, also release similar structures. The fatty acid chain signals from the mobile lipids, observable in the H NMR spectrum, and the fluorescence polarization of the DPH probe are strongly affected by RNAase digestion, thus indicating an association between RNA molecules and lipids. The enzymatic marker cytochrome c reductase was assayed to rule out possible contamination from endoplasmatic reticulum. A high alkaline phosphatase activity was instead found in the supernatant samples, thus indicating that the shed material is released by the plasma membrane.

Erythrocyte Na-K-Cl cotransport activity in low renin essential hypertensive patients. A 23Na nuclear magnetic resonance study.

The Na-K-Cl cotransport activity in red blood cells from essential hypertensive men with low (n = 8, mean age 42 +/- 4 years) or normal renin activity (n = 4, mean age 43 +/- 3 years), and in normotensive men with normal renin activity (n = 7, mean age 38 +/- 4 years) has been evaluated by means of a recently developed 23Na nuclear magnetic resonance (NMR) method. Sodium efflux was determined by relating the resonating frequency of the NMR signal from extracellular sodium to sodium concentration in the presence of the shift reagent Dy(PPP)2(7-). The maximum Na+ efflux driven by cotransport (Vmax) was measured in Na(+)-loaded erythrocytes in the presence of ouabain to block the Na-K-Cl pump activity. A significant difference (P < 0.05) was found in Vmax values of low renin patients (0.70 mmol/h/L cells, range 0.40 to 0.90 mmol/h/L cells) as compared with normotensive controls (0.39 +/- 0.08 mmol/h/L cells) and normal renin hypertensives (mean 0.49 +/- 0.04 mmol/h/L cells). In conclusion, this study showed an increased activity of the Na-K-Cl cotransport in red blood cells from low renin hypertensive men as compared with normal renin hypertensives and normotensives.

Multiexponential T2 relaxation in Fricke agarose gels: implications for NMR dosimetry.

NMR relaxation times T1 and T2 of agarose and Fricke agarose gels have been measured in the range 17-51 MHz. The analysis of the spin echo curves indicates a multiexponential behaviour, characterized by three components, at all the examined frequencies. The relative T2 values, ranging from a few to a hundred milliseconds, can be attributed to different species of water molecules present in the gel. Two of these components are characterized by relaxation rates R2a and R2b, more sensitive than R1 to gamma irradiation, the sensitivity S being S(R1) = 0.066 s-1 Gy-1, S(R2a) = 0.088 s-1 Gy-1, S(R2b) = 0.17 s-1 Gy-1. The three T2 values decrease as a function of frequency, but no gain in dose sensitivity is obtained by changing the working frequency in the examined range. The relaxivity of agarose gels containing ferrous or ferric ions has also been measured and found to be different from those of the corresponding solutions in the absence of agarose. Thus it was possible to estimate the irradiation yield from three independent parameters, R1, R2a and R2b. No effect of the dose rate or of the source energy has been observed for any of these parameters.

Bayesian estimation of relaxation times T(1) in MR images of irradiated Fricke-agarose gels.

The authors present a novel method for processing T(1)-weighted images acquired with Inversion-Recovery (IR) sequence. The method, developed within the Bayesian framework, takes into account a priori knowledge about the spatial regularity of the parameters to be estimated. Inference is drawn by means of Markov Chains Monte Carlo algorithms. The method has been applied to the processing of IR images from irradiated Fricke-agarose gels, proposed in the past as relative dosimeter to verify radiotherapeutic treatment planning systems. Comparison with results obtained from a standard approach shows that signal-to noise ratio (SNR) is strongly enhanced when the estimation of the longitudinal relaxation rate (R1) is performed with the newly proposed statistical approach. Furthermore, the method allows the use of more complex models of the signal. Finally, an appreciable reduction of total acquisition time can be obtained due to the possibility of using a reduced number of images. The method can also be applied to T(1) mapping of other systems.

Dose distribution of proton beams with NMR measurements of Fricke-agarose gels.

Fricke-agarose gels have been irradiated with a proton beam. Then samples have been extracted at different depths with respect to the beam penetration distance, corresponding to different irradiation doses. Relaxation times T1 and T2, measured at 17 MHz, appear sensitive to this kind of radiation. In particular, T2 exhibits three components T2a, T2b and T2c, the first two being sensitive to proton irradiation. At 1% agarose concentration, the relaxation rates R1 = 1/T1, R2a = 1/T2a and R2b = 1/T2b of samples irradiated with both modulated and unmodulated beams, increase with the dose, irrespective of the beam energy. The yield G of Fe3+ ions per 100 eV of absorbed energy is always higher than that obtained for gamma irradiated samples.

Changes in soluble metabolites induced in tumour cells by gamma rays and proton beams: a 1H magnetic resonance spectroscopy study.

Cultured HeLa cells have been irradiated with a single acute dose of either gamma rays (40 Gy) or low energy proton beams (20 Gy). 1H magnetic resonance spectra of perchloric acid extracts (PCA) prepared at different times after irradiation show an intensity decrease of signal from glutathione and a parallel increase of lactate signal. Intensity ratios of signals of glycerophosphorylcholine to that of phosphorylcholine increase with time after irradiation. Irradiating cells with gamma rays or proton beams at half a dose obtain effects of comparable extent. The present experiments indicate that relevant metabolic alterations take place in cells damaged by irradiation. Finally, these data represent a first indication that the relative biological efficiency of low energy proton beams with respect to gamma rays can reach a value of 2 as evidenced by cell metabolic impairment.

Dose reconstruction in irradiated Fricke-agarose gels by means of MRI and optical techniques: 2D modelling of diffusion of ferric ions.

Fricke-agarose gels have elicited much interest in the field of radiation dosimetry, as tissue-equivalent dosemeters. magnetic resonance (MR) relaxation rates are measured for dose reconstruction. A major problem of Fricke-agarose gels is the diffusion of the ferric ions formed after irradiation. Knowledge of the diffusion coefficient of ferric ions may be necessary. Xylene orange, a dye that specifically chelates ferric ions, was added to the Fricke gel system to reduce ion diffusion and, as the binding gives a coloured complex, to allow optical detection of ferric ions. Diffusion of ferric ions in two dimensions and time evolution of ion concentration were evaluated. MR images were obtained at different times after irradiation. Ferric ion distribution, the corresponding images and the doses at different times after irradiation were reconstructed taking into account the calculated diffusion coefficients. Diffusion was then estimated by means of two different optical methods. The agreement obtained supports the reliability of the MRI method and of the model.

Biochemical and NMR studies on structure and release conditions of RNA-containing vesicles shed by human colon adenocarcinoma cells.

Large amounts of particulate material, mostly lipid vesicles, are released by human colon adenocarcinoma HCT-8R cells when they are packed at high density in saline solution. RNA is also present in the released structures. Vesicle sheding is displayed only by healthy and viable cells. The process, in our experimental conditions, lasts up to 40 min, and can be restored by supplementing cells with nutrients and oxygen. RNA and lipids give rise to IH and 3IP NMR signals. The process is somehow related to a thermotropic transition observed by means of IH NMR spectroscopy for peculiar lipid domains in the plasma membrane. Analysis of 3IP NMR spectra of the phosphodiester groups, upon pH variation, indicates strong interaction between RNA and proteins in an assembled structure. A constant amount of polyA+ RNA can be recovered from the vesicles. The electrophoretic pattern and in vitro protein synthesis indicate that mRNA can be isolated as a functionally active molecule with a major 5 Kb fraction.

1H-MRS lipid signal modulation and morphological and ultrastructural changes related to tumor cell proliferation.

Changes in signal intensity of (1)H-MRS mobile lipids, mostly triglycerides, have been observed in cultured HeLa cells during exponential growth. Signals were intense in the first days after seeding and almost disappeared after a few days in culture. Choline-based metabolites were not affected by growth. Treatment with the antitumor drug lonidamine, which blocks cell proliferation and cell progression through cycle, increased lipid signal intensity. Morphological changes in the organization of the cell surface were detected by scanning electron microscopy during exponential growth, and were confirmed by freeze fracture analysis. The observed metabolic and structural modifications during cell growth were correlated to cell cycle progression of HeLa cells, as high-intensity lipid signals were typical of cells with a high percentage of S- and( )G(2) + M-phases, while cells with a high frequency in G(1)-phase were characterized by mobile lipid signals of very low intensity. Magn Reson Med 42:248-257, 1999.