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.

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.

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.

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.

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.

MRS study of the interaction of dihydropyridines with lipid molecules in phosphatidylcholine vesicles.

Dihydropyridines (DHPs), synthetic molecules used as antihypertensive agents, bind to plasma membrane receptors following diffusion through the hydrophobic phase. In this study, MRS technique has been used to clarify the interactions of the dihydrophyridines Nifedipine and Lacidipine within the lipid bilayer. 1D and 2D 1H MRS at high field have been employed to examine the behavior of unilamellar dimyristoyl-phosphatidylcholine liposomes when the two drugs have been inserted in the bilayer. In particular, the study represents an innovative application of 2D 1H NOESY technique to clarify different mechanisms of interactions of small molecules inside model membranes. On the other hand, 31P measurements have been performed in multilamellar dimyristoyl-phosphatidylcholine lipsomes to detect alterations of lipid polymorphic phases. The experiments show that the two dihydropyridines interact with the lipids by different modalities. Lacidipine undergoes a very strong interaction with lipids, possibly inducing a phase segregation of lipid molecules into the bilayer, while self-association seems to be the prevalent interaction of Nifedipine inside the bilayer.

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.

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.

Continuous quality improvement in radiotherapy: the role of the "Istituto Superiore di Sanità" in its application over the national territory.

The "Laboratorio di Fisica" of the "Istituto Superiore di Sanità" has undertaken some initiatives to tackle the problem of patients' radiation protection in relation to the issues of radiotherapy, both with respect to the promulgation of new National and European laws and directives and to the extremely rapid and innovative technological developments involved in this field. First, interdisciplinary study groups were organized to identify the aspects on which recommendations to be applied at the national level could be based, and to provide for their development. Second, a national plan was launched for continuous quality improvement in radiotherapy, intended as a program of controls and correctives implemented in radiotherapy to ensure its compliance with predefined standards together with its continuous improvement. Within this plan, a group of experts coordinated by the "Istituto Superiore di Sanità" has promoted two different intercomparisons of dosimetry to evaluate the differences between the measured dose and the calculated dose 1) with reference to a water phantom and 2) during a simulated treatment of rectal cancer in a plastic phantom.

Radiation effects on soluble metabolites in cultured HeLa cells examined by 1H MRS: changes in concentration of glutathione and of lipid catabolites induced by gamma rays and proton beams.

Cultured HeLa cells were irradiated with a single acute dose of either gamma rays (40 Gy) or low-energy proton beams (20 Gy). (1)H magnetic resonance spectra of intact cells harvested at different times after irradiation and of the correspondent perchloric acid (PCA) extracts prepared at different times after irradiation were run. Selected signals from glutathione and lactate were examined with the aim of investigating effects of irradiation on antioxidative stores and on mitochondrial activity. An increase of signal intensity of glutathione (GSH) takes place at 15 and 24 hr after irradiation, while a decrease of its signal intensity, accompanied by an increase of that of free glutamate, starts appearing 48 hr after irradiation. Lactate signal increases 48 hr after irradiation. Signals from lipid catabolites were also examined to explore their sensitivity in predicting the response to radiotherapy. Intensity ratios of signals of glycerophosphorylcholine and choline to that of phosphorylcholine increase with time after irradiation. Irradiating cells with gamma rays or proton beams at half a dose produces effects comparable to the metabolic variations presented here. The present experiments allow more insight into the complex pattern of the changes of GSH by irradiation and indicate that magnetic resonance spectroscopy signals from GSH, glutamate, lactate, and lipid catabolites are affected by irradiation. Finally, these data represent a first indication that the relative biological efficiency for some metabolic damage of low-energy proton beams with respect to gamma rays can reach a value of 2.

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.

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.

Characterization of a phospholipid adduct formed in Sprague Dawley rats by chloroform metabolism: NMR studies.

The formation of a covalent adduct to a single phospholipid by the oxidative chloroform metabolite, phosgene, is demonstrated in liver mitochondria of phenobarbital-pretreated Sprague Dawley (SD) rats treated with CHCl3. The densitometric analysis of the phosphorus stained extracted phospholipids showed that the formation of this adduct in liver mitochondria is accompanied by a decrease of phosphatidylethanolamine and cardiolipin. The characterization of this adduct was performed with a multinuclear NMR approach by comparison with the decreased phospholipids. Treatment of rats with [13C]chloroform resulted in an intense 13C NMR peak from either an esteric or amidic carbonyl. Very strong similarities in fatty acid composition were found between phosphatidylethanolamine and the phosgene-modified PL, using 13C and 1H NMR spectroscopy. A multiplet at 3.91 ppm coupled to a signal at 3.41 ppm was shown by two-dimensional 1H NMR in the adduct spectrum. This cross peak was interpreted as arising from the shifted resonances of the two PE head group methylene groups, due to the binding with phosgene. 31P spectrum of the adduct was identical to that of phosphatidylethanolamine. We concluded that the chloroform adduct is a modified phosphatidylethanolamine, with the phosgene-derived carbonyl bound to the amine of the head group.

Hormonally induced modulation in the phosphate metabolites of breast cancer: analysis of in vivo 31P MRS signals with a modified prony method.

A modified Prony method (MPM) was applied to analyze the main signals present in spatially resolved 31P NMR spectra of MCF7 breast tumors implanted in nude mice. First, the method was tested on synthetic data to establish its limits of reliability. Its performance with respect to peak identification and quantification of signal intensities was then exploited on data from three implanted tumors during hormonal manipulation with estrogen and the antiestrogenic drug tamoxifen. The phosphomonoester peak was resolved into phosphocholine (PC) and phosphoethanolamine (PE). Treatment with tamoxifen led to a significant reduction in the PE to PE+PC peak amplitude ratio in the tumors under consideration. MPM analysis also revealed the presence of two different inorganic phosphate pools: a larger acidic pool and a smaller alkaline pool during estrogen-induced growth and the reverse during tumor regression.

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.

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.

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.