Abstract: High-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy was used to examine and compare the metabolic variations that occur in cells of the HL60 promyelocytic leukemia cell line after induction of apoptosis by ionizing radiation and the antineoplastic drug doxorubicin as well as after induction of necrosis by heating. Apoptosis and necrosis were confirmed by fluorescence microscopy using the chromatin stain Hoechst 33258, agarose gel electrophoresis of DNA, and determination of caspase 3 enzymatic activity. The 1H-NMR experiments revealed that the spectra of both samples containing apoptotic cells were characterized by the same trend of several important metabolites. Specifically, an increase in CH2 and CH3 mobile lipids, principally of CH2, decreases in glutamine and glutamate, choline-containing metabolites, taurine and reduced glutathione were observed. By contrast, the sample containing necrotic cells presented a completely different profile of 1H-NMR metabolites since it was characterized by a significant increase in all the metabolites examined, with the exception of CH2 mobile lipids, which remain unchanged, and reduced glutathione, which decreased. The results suggest that variations in 1H-NMR metabolites are specific to apoptosis independent of the physical or chemical nature of the stimulus used to induce this mode of cell death, while cells dying from necrosis are characterized by a completely different behavior of the same metabolites.
Abstract: The metabolic changes that occur in MG-63 osteosarcoma three-dimensional tumor spheroids exposed to 2 Gy of ionizing radiation, a dose that is comparable to radiation therapy, were studied using high-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. Specifically, the (1)H-NMR spectra of control and exposed MG-63 spheroids were compared. Small spheroids (about 50-80 microm in diameter) with no hypoxic center were used. The spectra of whole MG-63 spheroids as well as the perchloric acid extracts of these systems were evaluated. Cell damage was also examined by lactate dehydrogenase release and changes in cell growth. No cell damage was observed, but numerous metabolic changes took place in spheroids after exposure to ionizing radiation. In particular, significant increases in both CH(2) and CH(3) mobile lipids, considered by many authors as markers of apoptosis and also present in MG-63 spheroids undergoing overt apoptosis, were observed in spheroids irradiated with 2 Gy. However, the chromatin dye Hoechst 33258 and DNA fragmentation assays showed no overt apoptosis up to 7 days after irradiation with this low dose. Thus it is evident that increases in mobile lipids do not always indicate actual cell death. A detailed analysis of the other metabolic changes observed appears to suggest that the cell death program was initiated but not completed. In fact, the completely different behavior of two important cellular defense mechanisms, reduced glutathione and taurine, in spheroids irradiated with 2 Gy and in those undergoing overt apoptosis seems to indicate that these systems are protecting spheroids from actual cell death. In addition, these data also suggest that (1)H-NMR can be used to examine the effects of low doses of ionizing radiation in spheroids, a cell model of great complexity that closely resembles tumors in vivo. The importance of this possibility in relation to reaching the ultimate goal of a better evaluation of the outcome of radiotherapy protocols should not be ignored.
Abstract: The metabolic changes that occur as a function of time in MG-63 osteosarcoma three-dimensional tumor spheroids undergoing radiation-induced apoptosis were studied using high-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. Specifically, the (1)H-NMR spectra of MG-63 spheroids collected at 24, 48 and 72 h after exposure to 5 Gy of ionizing radiation were compared to the spectra of their respective controls. Small spheroids (about 50-80 microm in diameter) with no hypoxic center were used. Apoptosis was verified by both staining of spheroid DNA with the Hoechst 33258 dye and determination of caspase 3 enzyme activity at the three times examined. The results demonstrate that, as the percentage of apoptosis rises with time after exposure to ionizing radiation, the metabolic changes that take place in MG-63 spheroids follow very precise temporal dynamics. In particular, significant time-related increases in both CH(2) and CH(3) mobile lipids, considered by many authors as markers of apoptosis, were observed. In addition, temporal variations were also observed in choline-containing metabolites, reduced glutathione (GSH), glutamine/glutamate, taurine, alanine, creatine/phosphocreatine and lactate. These data show that in addition to CH(2) and CH(3) lipids, other metabolites can also be extremely useful in a deeper understanding of the temporal dynamics of radiation-induced apoptosis. This comprehension is particularly important in spheroids, a cell model of great complexity that resembles in vivo tumors much more closely than monolayer cultures. Ultimately, it is hoped that such studies can help to evaluate the outcome of radiotherapy protocols more accurately.
Abstract: High resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy was used to examine the response of the MG-63 osteosarcoma cell line grown in monolayer and as 3-dimensional tumor spheroids to the same low dose (2 Gy) of ionizing radiation. The MG-63 cells and spheroids were irradiated at 24 h of growth and the 1H-NMR spectra of whole control and irradiated monolayer cells and of whole control and irradiated multicellular spheroids collected after another 24 h were compared. The 1H-NMR spectra of the perchloric acid extracts as well as the 2-dimensional 1H-NMR spectra of both pairs of cell systems were also obtained. Possible radiation-induced cell damage was determined by lactate dehydrogenase (LDH) release and variations in cell growth, while cell death was evaluated by chromatin dye Hoechst staining and DNA fragmentation assays. The results demonstrated that no cell damage took place, but that significant variations in numerous metabolites occured in both the monolayer cells and the spheroids after irradiation. Most of the changes observed were very similar in nature. In fact, significant increases in lactate, alanine, creatine and phosphocreatine and choline-containing metabolites and a significant decrease in glutathione (GSH) were observed in both cells and spheroids. However, while significant increases in CH2 and CH3 mobile lipids, glutamine/glutamate, taurine and inositol were seen in the spheroids, no variations in CH2 or CH3 lipids, glutamine/glutamate or taurine were recorded in the MG-63 cells grown in monolayer after irradiation. In addition, a significant decrease rather than a significant increase in inositol was also noted in the monolayer cells. The data presented seem to suggest that, although neither monolayer cells nor spheroids show apparent signs of damage after exposure to the same dose of ionizing radiation, very different cell death responses as well as very diverse antioxidant/osmoregulatory reactions were triggered by this stressing agent.
Abstract: PURPOSE: To study cell damage and possible apoptosis in K562 human erythroleukemic cells exposed for 2 h to an extremely low frequency (ELF) 50 Hz sinusoidal magnetic field with a magnetic induction of either 1 or 5 mT using high resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. MATERIALS AND METHODS: One-dimensional 1H-NMR spectra were obtained on whole K562 cells and perchloric acid extracts of these cells. In addition, two-dimensional 1H-NMR spectra were also acquired. Cell damage was examined by lactate dehydrogenase release and changes in cell growth were monitored by growth curve analyses, bromodeoxyuridine incorporation and Ki67 antigen localization. Cell death (necrosis and apoptosis) were also studied by using the chromatin dye Hoechst 33258. RESULTS: The variations in numerous metabolites observed with 1H-NMR reveal apoptosis-like behavior in response of K562 cells to ELF fields. CONCLUSION: 1H-NMR can be extremely useful in studying the effects of ELF fields on cells. In particular, the variations in metabolites which suggest apoptosis-like behavior occur when the cells are not identifiable as apoptotic by more traditional techniques.
Abstract: Because of the association between inhalation of airborne particulate matter (PM) and human respiratory and cardiovascular disease, it is necessary to understand the tissue damage induced by these particles. One of the cell types principally involved in the body's reaction to PM are macrophages, which remove particles in the airway passages and the lungs through phagocytosis. In fact, when macrophages are exposed to a toxic agent such as PM, they undergo a series of changes (including variations in morphology, an increase in glycolysis, and consequent lactate production and the release of cytokines such as interleukin-6 and tumor necrosis factor-alpha) necessary to transform them from "resting" to "activated" macrophages. Because (1)H NMR is extremely useful in monitoring, noninvasively, macrophage metabolism and because this technique has never been utilized to examine macrophage activation after exposure to PM, it was the purpose of the present study to investigate the effects of PM exposure on the RAW 264.7 stabilized macrophage cell line using (1)H NMR spectroscopy. PM with a diameter <2.5 microm (PM 2.5) was utilized because a closer association to mortality and adverse respiratory health effects has been found with this fraction than with particles of a larger size. Measurements were conducted on whole cells at both 500 and 700 MHz as well as on perchloric acid extracts at 700 MHz. Significant variations in numerous metabolites were seen at very low concentrations of PM 2.5. Many of these changes point to activation of RAW 264.7 macrophages even at doses of PM 2.5 much lower than those commonly employed in cell studies. These results are particularly significant since the same concentrations of PM did not induce changes in morphology and release of cytokines in these cells. Therefore, (1)H NMR spectroscopy is an extremely sensitive probe in observing subtle variations in macrophages after exposure to PM 2.5.
Abstract: High resolution proton nuclear magnetic resonance ((1)H NMR) spectroscopy was used to determine if the same cell line (MG-63 human osteosarcoma cells) grown in monolayer or as small (about 50-80 microm in diameter), three-dimensional tumor spheroids with no hypoxic center has different metabolic characteristics. Consequently, the (1)H NMR spectra were obtained from both types of cultures and then compared. The results indicate that the type of cellular spatial array determines specific changes in MG-63 cells. In particular, small but significant differences in lactate and alanine indicating a perturbation in energy metabolism were observed in the two cell models. In addition, although variations in CH(2) and CH(3) groups were also seen, it is not possible at this time to establish if lipid metabolism is truly different in cells and spheroids.
Abstract: In this paper a new time-domain frequency-selective quantification algorithm is presented. Frequency-selective quantification refers to a method that analyzes spectral components in a selected frequency region, ignoring all the other components outside. The algorithm, referred to as MeFreS (Metropolis Frequency-Selective), is based on rank minimization of an opportune Hankel matrix. The minimization procedure is satisfied by the down-hill simplex method, implemented with the simulated annealing method. MeFreS does not use any preprocessing step or filter to suppress nuisance peaks, but the signal model function is directly fitted. In this manner, neither inherent signal distortions nor estimation biases to be corrected occur. The algorithm was tested with Monte Carlo simulations. A comparison with VARPRO and AMARESw algorithms was carried out. Finally, two samples of known content from NMR data were quantified.
Abstract: The high resolution proton nuclear magnetic resonance (1H-NMR) spectra of two different cell lines exhibiting multidrug resistance (MDR) as demonstrated by the expression of the well-known energy-driven, membrane-bound 170 kDa P-glycoprotein pump known as Pgp were investigated. In particular, the mobile lipid (ML) profile, and the growth and biochemical characteristics of MCF-7 (human mammary carcinoma) and LoVo (human colon adenocarcinoma) sensitive and resistant tumor cells were compared. The results indicate that both MCF-7 and LoVo resistant cells have a higher ML intensity than their respective sensitive counterparts. However, since sensitive and resistant cells of each pair grow in the same manner, variations in growth characteristics do not appear to be the cause of the ML changes as has been suggested by other authors in non-resistant tumor cells. In order to investigate further the origin of the ML changes, lipid analyses were conducted in sensitive and resistant cell types. The results of these experiments show that resistant cells of both cell types have a greater amount of esterified cholesterol and saturated cholesteryl ester and triglyceride fatty acid than their sensitive counterparts. From a thorough analysis of the data obtained in this paper utilizing numerous techniques including biological, biophysical and biochemical ones, it is hypothesized that cholesterol and triglyceride play a pivotal role in inducing changes in NMR ML signals. The importance of these lipid variations in MDR is discussed in view of the controversy regarding the origin of ML signals and the paramount role played by the Pgp pump in resistance.
Abstract: Recently, a new method for quantitatively comparing NMR spectra of control and treated samples, in order to examine the possible occurring variations in cell metabolism and/or structure in response to numerous physical, chemical, and biological agents, was proposed. This method is based upon the utilization of the maximum superposition normalization algorithm (MaSNAl) operative in the frequency domain and based upon maximizing, by an opportune sign variable measure, the spectral region in which control and treated spectra are superimposed. Although the frequency-domain MaSNAl algorithm was very precise in normalizing spectra, it showed some limitations in relation to the signal-to-noise ratio and to the degree of diversity of the two spectra being analyzed. In particular, it can rarely be applied to spectra with a small number of visible signals not buried in the noise such as generally in vivo spectra. In this paper, a time-domain normalization algorithm is presented. Specifically, it consists in minimizing the rank of a Hankel matrix constructed with the difference of the two free induction decay signals. The algorithm, denoted MiRaNAl (minimum rank normalization algorithm), was tested by Monte Carlo simulations as well as experimentally by comparing two samples of known contents both with the new algorithms and with an older method using a standard. Finally, the algorithm was applied to real spectra of cell samples showing how it can be used to obtain qualitative and quantitative biological information.
