Metabolite changes in BT4C rat gliomas undergoing ganciclovir-thymidine kinase gene therapy-induced programmed cell death as studied by 1H NMR spectroscopy in vivo,ex vivo,and in vitro

Programmed cell death was induced by HSV-tk gene therapy in rat BT4C glioma cells, and metabolite changes associated with cell damage were monitored in vivo by 1H NMR spectroscopy and ex vivo by high resolution magic angle spinning (HRMAS) 1H NMR, and in vitro in perchloric acid extracts of tumors. Metabolite concentrations, as quantified in vivo using water as an internal reference and in vitro in extracts, were correlated with cell density. The results showed that both in vivo and in vitro glycine and creatine concentrations followed volume-averaged cell density, whereas that of total choline-containing compounds was unaffected by a cell loss approaching 60%. Meanwhile, both saturated and unsaturated 1H NMR visible lipids increased. HRMAS 1H NMR spectroscopy of the tumor samples at 14.1 tesla demonstrated the presence of nucleotide peaks from adenosine and uridine nucleotides in glioma samples ex vivo. The assignment of a doublet at 7.95 ppm to UDP was confirmed by spiking experiments of tumor extracts in conjunction with 1H and 31P NMR spectroscopy. HRMAS also resolved the choline-containing peak at 3.2 ppm in vivo into resonances from choline (3.20 ppm), phosphocholine (3.22 ppm), glycerophosphocholine (3.24 ppm), and taurine (3.26 ppm). These resonances were uncorrelated with temporal progression through programmed cell death. Our results show that 1H NMR-detected lipids and some of the small molecular weight metabolites respond to gene therapy. However, the choline-containing compounds are unaffected by severe decline in cell density. The latter observation supports the idea that triacylglycerols, rather than membrane phospholipids, are the key components of 1H NMR visible lipids, and it also casts doubt on the validity of resonance of choline-containing compounds as a diagnostic marker of programmed cell death in vivo.     

Effects of Alkaline Phosphatase Activity on Nucleotide Measurements in Aquatic Microbial Communities

Alkaline phosphatase (APase) activity was detected in aquatic microbial assemblages from the subtropics to Antarctica. The occurrence of APase in environmental nucleotide extracts was shown to significantly affect the measured concentrations of cellular nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine triphosphate, uridine triphosphate, and cytidine triphosphate), adenylate energy charge, and guanosine triphosphate/adenosine triphosphate ratios, when conventional methods of nucleotide extraction were employed. Under the reaction conditions specified in this report, the initial rate of hydrolysis of adenosine triphosphate was directly proportional to the activity of APase in the sample extracts and consequently can be used as a sensitive measure of APase activity. A method was devised for obtaining reliable nucleotide measurements in naturally occurring microbial populations containing elevated levels of APase activity. The metabolic significance of APase activity in microbial cells is discussed, and it is concluded that the occurrence and regulation of APase in nature is dependent upon microscale inorganic phosphate limitation of the autochthonous microbial communities.     

Insulin inhibits changes in the phospholipid profiles in sciatic nerves from streptozocin-induced diabetic rats: A phosphorus-31 magnetic resonance study

Sciatic nerve phospholipids obtained from insulin-treated streptozocin-induced diabetic, non-treated streptozocin-induced diabetic, and healthy, control male Sprague-Dawley rats after eighteen weeks of diabetes were studied by ³¹P NMR spectrometry. Eleven phospholipids resonances were identified as follows: Phosphatidic acid (Chemical shift, 0.30 ppm), dihydrosphingomyelin (0.13 ppm), ethanolamine plasmalogen (0.07 ppm), phosphatidylethanolamine (0.03 ppm), phosphatidylserine (−0.05 ppm), sphingomyelin (−0.09 ppm), lysophosphatidylcholine (−0.28 ppm), phosphatidylinositol (−0.30 ppm), alkylacylglycerophosphorylcholine (−0.78 ppm), choline plasmalogen (−0.80 ppm), and phosphatidylcholine (−0.84 ppm). Diabetic rats showed that phosphatidylcholine was significantly elevated p > 0.05, and ethanolamine plasmalogen and choline plasmalogen were significantly lower when compared with both control and insulin treated rats. The choline ratio (choline-containing phospholipids over noncholine phospholipids) was significantly elevated in the diabetic group, when compared with both control and insulin-treated groups. The ethanolamine ratio (ethanolamine-containing phospholipids over nonethanolamine phospholipids) and the ratio of the ethanolamine ratio over the choline ratio, was significantly elevated in the control and the insulin-treated groups when compared with the diabetic group. The presence of phosphatidic acid and the significance in phosphatidylcholine and ethanolamine plasmalogen, suggested that insulin had a role in the phosphatidylcholine metabolism in the rat nerve.     

The mechanism of galactosamine toxicity revisited; a metabonomic study

1H NMR spectroscopy was used to investigate the metabolic effects of the hepatotoxin galactosamine (galN) and the mechanism by which glycine protects against such toxicity. Rats were acclimatized to a 0 or 5% glycine diet for 6 days and subsequently administered vehicle, galN (500 mg/kg), glycine (5% via the diet), or both galN and glycine. Urine was collected over 12 days prior to administration of galN and for 24 hours thereafter. Serum and liver tissue were sampled on termination, 24 hours post-dosing. The metabolic profiles of biofluids and tissues were determined using high-field 1H NMR spectroscopy. Orthogonal-projection to latent structures discriminant analysis (O-PLS-DA) was applied to model the spectral data and enabled the hepatic, urinary, and serum metabolites that discriminated between control and treated animals to be determined. Histopathological data and clinical chemistry measurements confirmed the protective effect of glycine. The level of N-acetylglucosamine (glcNAc) in the post-dose urine was found to correlate strongly with the degree of galN-induced liver damage, and the urinary level of glcNAc was not significantly elevated in rats treated with both galN and glycine. Treatment with glycine alone was found to significantly increase hepatic levels of uridine, UDP-glucose, and UDP-galactose, and in view of the known effects of galactosamine, this suggests that the protective role of glycine against galN toxicity might be mediated by changes in the uridine nucleotide pool rather than by preventing Kupffer cell activation. Thus, we present a novel hypothesis: that administration of glycine increases the hepatic uridine nucleotide pool which counteracts the galN-induced depletion of these pools and facilitates complete metabolism of galN. These novel data highlight the applicability of NMR-based metabonomics in elucidating multicompartmental metabolic consequences of toxicity and toxic salvage.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. I. Kinetics of incorporation of nucleosides into nucleotide pools and pool sizes during growth cycle

Results from kinetic studies on the incorporation of ³H-5-uridine and ³H-8-adenosine into the acid-soluble nucleotide poor and nucleic acids by Novikoff hepatoma cells (subline N1S1-67) in suspension culture indicate that the uridine transport reaction is saturated at about 100 μM and that for adenosine at about 10 μM nucleoside in the medium, and that above 100 μM simple diffusion becomes the predominant mode of entry of both nucleosides into the cell. The Km of the transport reactions is approximately 1.3 × 10^?5 M for uridine and 6 × 10^?6 M for adenosine. The incorporation of these nucleosides into both the nucleotide pool and into nucleic acids seems to be limited by the rate of entry of the nucleic acid synthesis from the rate of incorporation of nucleosides. Other complicating factors are a change with time of labeling in the relative proporation of nucleoside incorporated into DNA and into the individual nucleotides of RNA, the splitting of uridine to uracil by th ecells, the deamination of adenosine kto inosine and the subsequent cleavage of inosine to hypoxanthine. Various lines of evidence are presented which indicate that the overall nucleotide pools of the cells are very small under normal growth conditions. During growth in the presence of 200 μM uridine or adenosine, however, the cells continue to convert the nucleosides into intracellular nucleotides much more rapidly than required for nucleic acid synthesis. This results in an accumulation of free uridine and adenosine nucleotides in the cells, the maximum amounts of which are at least equivalent to the amount of these nucleotides in total cellular RNA.     

Neuroblastoma Growth Factors Derived from Neurofibroma (NF1): Participation of Uridine in a Neuroblastoma Growth

Abstract: Human glioma cell extracts were found to elicit a marked growth-promoting activity on human neuroblastoma cells. This activity was also detected in the extracts of neurofibroma type 1 (NF1; von Recklinghausen neurofibromatosis) comprising aberrant Schwann cell growth. The purified substance from the NF1 extracts by HPLC on ODS columns was identical to a pyrimidine nucleoside, uridine, the chemical structure of which was identified by gas chromatography-mass spectrometry. The authentic uridine showed a strong growth-promoting activity on human neuroblastoma cells. Other purine or pyrimidine nucleotides, their derivatives, and ribose sources for their syntheses were employed to test the activity; a purine nucleoside, adenosine, showed a stronger activity than uridine. The current study raises the possibility that human neuroblastoma cells may be affected by dysfunctions of the de novo pathway of both purine and pyrimidine nucleotide biosyntheses.     

15N-labeled tRNA. Identification of 4-thiouridine in Escherichia coli tRNASer1 and tRNATyr2 by 1H-15N two-dimensional NMR spectroscopy

Uridine is uniquely conserved at position 8 in elongator tRNAs and binds to A14 to form a reversed Hoogsteen base pair which folds the dihydrouridine loop back into the core of the L-shaped molecule. On the basis of 1H NMR studies, Hurd and co-workers (Hurd, R. E., Robillard, G. T., and Reid, B. R. (1977) Biochemistry 16, 2095-2100) concluded that the interaction between positions 8 and 14 is absent in Escherichia coli tRNAs with only 3 base pairs in the dihydrouridine stem. We have taken advantage of the unique 15N chemical shift of N3 in thiouridine to identify 1H and 15N resonances for the imino units of S4U8 and s4U9 in E. coli tRNASer1 and tRNATyr2. Model studies with chloroform-soluble derivatives of uridine and 4-thiouridine show that the chemical shifts of the protons in the imino moieties move downfield from 7.9 to 14.4 ppm and from 9.1 to 15.7 ppm, respectively; whereas, the corresponding 15N chemical shifts move downfield from 157.5 to 162.5 ppm and from 175.5 to 180.1 ppm upon hydrogen bonding to 5'-O-acetyl-2',3'-isopropylidene adenosine. The large difference in 15N chemical shifts for U and s4U allows one to unambiguously identify s4U imino resonances by 15N NMR spectroscopy. E. coli tRNASer1 and tRNATyr2 were selectively enriched with 15N at N3 of all uridines and modified uridines. Two-dimensional 1H-15N chemical shift correlation NMR spectroscopy revealed that both tRNAs have resonances with 1H and 15N chemical shifts characteristic of s4UA pairs. The 1H shift is approximately 1 ppm upfield from the typical s4U8 resonance at 14.8 ppm, presumably as a result of local diamagnetic anisotropies. An additional s4U resonance with 1H and 15N shifts typical of interaction of a bound water or a sugar hydroxyl group with s4U9 was discovered in the spectrum of tRNATyr2. Our NMR results for tRNAs with 3-base pair dihydrouridine stems suggest that these molecules have an U8A14 tertiary interaction similar to that found in tRNAs with 4-base pair dihydrouridine stems.     

Calcium-dependent release of adenosine and uridine nucleotides from A549 cells

Extracellular nucleotides play an important role in lung defense, but the release mechanism and relative abundance of different nucleotide species secreted by lung epithelia are not well defined. In this study, to minimize cell surface hydrolysis, we used a low-volume, flow-through chamber and examined adenosine and uridine nucleotide concentrations in perfusate aliquots of human lung A549 cells challenged by 50% hypotonic shock. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (Ado) were quantified in high-performance liquid chromatography (HPLC) analysis of fluorescent etheno derivatives, and uridine triphosphate (UTP) and uridine diphosphate (UDP) were measured using HPLC-coupled radioenzymatic assays. After the onset of hypotonic shock, ATP, ADP, UTP, and UDP in the perfusates increased markedly and peaked at approximately 2.5 min, followed by a gradual decay in the next 15–20 min; peak changes in Ado and AMP were relatively minor. The peak concentrations and fold increment (in parentheses) were: 34±13 nM ATP (5.6), 11±5 nM ADP (3.7), 3.3±1.2 nM AMP (1.4), 23±7 nM Ado (2.1), 21 nM UTP (>7), and 11 nM UDP (27). Nucleotide release was almost completely abolished from cells loaded with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Under isotonic conditions, elevation of intracellular calcium with the calcium ionophore ionomycin (5 μM, 3 min) also released nucleotides with kinetics and relative abundance as above, albeit less robust. ADP:ATP (1:3) and UDP:UTP (1:2) ratios in perfusates from stimulated cells were markedly higher than the cytosolic ratios of these species, suggesting that a nucleotide diphosphate (NDP)-rich compartment, e.g., the secretory pathway, contributed to nucleotide release. Laser confocal microscopy experiments illustrated increased FM1-43 uptake into the plasma membrane upon hypotonic shock or ionomycin treatment, consistent with enhanced vesicular exocytosis under these conditions. In summary, our results strongly suggest that calcium-dependent exocytosis is responsible, at least in most part, for adenosine and uridine nucleotide release from A549 cells.     

Progression of programmed cell death in tobacco BY-2 cells is delineated by specific changes in de novo and salvage synthesis of purine nucleotides

Alterations in the pattern of purine nucleotide synthesis and degradation were investigated during programmed cell death (PCD) of tobacco BY-2 cells, induced by a simultaneous increase in the endogenous levels of nitric oxide (NO) and hydrogen peroxide. The de novo synthesis of purine nucleotides was estimated by following the metabolic fate of the [8-¹⁴C]5-aminoimidazole-4-carboxamide-1-β- d -ribofuranoside (AICAR), the salvage synthesis was investigated using [8-¹⁴C]adenine and adenosine, and the degradation pathway was studied by following the incorporation of [8-¹⁴C]inosine. The results indicated that specific changes in purine metabolism occurred during the death programme of tobacco cells. During the early phases of PCD, increases in the salvage activity of adenine and adenosine were observed, and these were related to the high activity of the two major salvage enzymes: adenine phosphoribosyltransferase (APRT) and adenosine kinase (ARK). During the following stages, a large fraction of purine nucleotide was also produced through the de novo pathway, suggesting a tight regulation between salvage and de novo synthesis. These changes were strictly associated with PCD, as they did not occur if NO or hydrogen peroxide was increased individually, or if actinomycin, which inhibits the death programme, was added to the medium in the presence of NO and hydrogen peroxide. These changes in purine nucleotide synthesis represent an early metabolic switch which may be needed to ensure the proper execution of all the high-energy demand processes characteristic of the death programme.     

Media supplementation for targeted manipulation of monoclonal antibody galactosylation and fucosylation

Monoclonal antibodies are critically important biologics as the largest class of molecules used to treat cancers, rheumatoid arthritis, and other chronic diseases. Antibody glycosylation is a critical quality attribute that has ramifications for patient safety and physiological efficacy—one that can be modified by such factors as media formulation and process conditions during production. Using a design-of-experiments approach, we examined the effect of 2-F-peracetyl fucose (2FP), uridine, and galactose on cell growth and metabolism, titer, and gene expression of key glycosylation-related proteins, and report how the glycoform distribution changed from Days 4 to 7 in a batch process used for IgG1 production from Chinese hamster ovary cells. We observed major glycosylation changes upon supplement addition, where the addition of 2FP decreased antibody fucosylation by up to 48%, galactose addition increased galactosylation by up to 21%, and uridine addition decreased fucosylation and increased galactosylation by 6% and 2%, respectively. Despite having major effects on glycosylation, neither galactose nor 2FP significantly affected cell culture growth, metabolism, or titer. Uridine improved peak cell densities by 23% but also reduced titer by ∼30%. The supplements caused significant changes in gene expression by Day 4 of the cultures where 2FP addition significantly reduced fucosyltransferase 8 and nucleotide sugar transporter gene expression (by ∼2-fold), and uridine addition significantly increased expression of UDP-GlcNAcT (SLC35A3) and B4GALT1–6 genes (by 1.5–3-fold). These gene expression data alongside glycosylation, metabolic, and growth data improve our understanding of the cellular mechanisms affected by media supplementation and suggest approaches for modifying antibody glycosylation in antibody production processes.     

Brassinolide-improved development of <Emphasis Type="Italic">Brassica napus</Emphasis> microspore-derived embryos is associated with increased activities of purine and pyrimidine salvage pathways

Cellular brassinolide (BL) levels regulate the development of Brassica napus microspore-derived embryos (MDEs). Synthesis and degradation of nucleotides were measured on developing MDEs treated with BL or brassinazole (BrZ), a biosynthetic inhibitor of BL. Purine metabolism was investigated by following the metabolic fate of ¹⁴C-labelled adenine and adenosine, substrates of the salvage pathway, and inosine, an intermediate of both salvage and degradation pathways. For pyrimidine, orotic acid, uridine and uracil were employed as markers for the de novo (orotic acid), salvage (uridine and uracil), and degradation (uracil) pathways. Our results indicate that utilization of adenine, adenosine, and uridine for nucleotides and nucleic acids increased significantly in BL-treated embryos at day 15 and remained high throughout the culture period. These metabolic changes were ascribed to the activities of the respective salvage enzymes: adenine phosphoribosyltransferase (EC 2.4.2.7), adenosine kinase (EC 2.7.1.20), and uridine kinase (EC 2.7.1.48), which were induced by BL applications. The BL promotion of salvage synthesis was accompanied by a reduction in the activities of the degradation pathways, suggesting the presence of competitive anabolic and catabolic mechanisms utilizing the labelled precursors. In BrZ-treated embryos, with depleted BL levels, the salvage activity of both purine and pyrimidine nucleotides was reduced and this was associated to structural abnormalities and poor embryonic performance. In these embryos, the activities of major salvage enzymes were consistently lower to those measured in their control (untreated) counterparts.     

Transport as the Rate-Limiting Step in the Incorporation of Uridine into Mengovirus Ribonucleic Acid in Novikoff Rat Hepatoma Cells

The incorporation of uridine into the nucleotide pool of actinomycin-treated, mengovirus-infected Novikoff rat hepatoma cells in culture follows simple Michaelis-Menten kinetics, and the apparent V(max) and K(m) values are similar to those for uridine transport by uninfected cells. Incorporation of uridine into mengovirus-specific ribonucleic acid (RNA) also follows Michaelis-Menten kinetics, and the apparent K(m) (about 10 mum) is approximately the same as for uridine transport. Inhibition of uridine transport by the presence of adenosine, persantin, or phenethyl alcohol inhibits simultaneously and to the same extent the incorporation of uridine into the nucleotide pool and into viral RNA, without affecting viral RNA synthesis per se. Phenethyl alcohol, however, also inhibits virus maturation. The inhibition of uridine incorporation into the nucleotide pool and into viral RNA is of the simple competitive type, indicating that transport into the cells is the rate-limiting step in the incorporation of uridine into mengovirus RNA. The results also indicate that treatment with actinomycin D or mengovirus infection does not affect uridine transport.     

Extracellular metabolism of nucleotides in neuroblastoma x glioma NG108-15 cells determined by capillary electrophoresis

1. The metabolism of extracellular nucleotides in NG108-15 cells, a neuroblastoma × glioma hybrid cell line, was studied by means of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC).2. In NG108-15 cells ATP, ADP, AMP, UTP, UDP, and UMP were hydrolyzed to the nucleosides adenosine and uridine indicating the presence of ecto-nucleotidases and ecto-phosphatases. The hydrolysis of the purine nucleotides ATP and ADP was significantly faster than the hydrolysis of the pyrimidine nucleotides UTP and UDP.3. ATP and UTP breakdown appeared to be mainly due to an ecto-nucleotide- diphosphohydrolase. ADP, but not UDP, was initially also phosphorylated to some extent to the corresponding triphosphate, indicating the presence of an adenylate kinase on NG108-15 cells. The alkaline phosphatase (ALP) inhibitor levamisole did not only inhibit the hydrolysis of AMP to adenosine and of UMP to uridine, but also the degradation of ADP and to a larger extent that of UDP. ATP and UTP degradation was only slightly inhibited by levamisole.4. These results underscore the important role of ecto-alkaline phosphatase in the metabolism of adenine as well as uracil nucleotides in NG108-15 cells. Dipyridamole, a potent inhibitor of nucleotide breakdown in superior cervical ganglion cells, had no effect on nucleotide degradation in NG108-15 cells.5. Dipyridamole, which is a therapeutically used nucleoside reuptake inhibitor in humans, reduced the extracellular adenosine accumulation possibly by allosteric enhancement of adenosine reuptake into the cells.     

Early biochemical and histological alterations in rat corticoencephalic cell cultures following metabolic damage and treatment with modulators of mitochondrial ATP-sensitive potassium channels

The present study was aimed at characterizing alterations of the nucleotide content and morphological state of rat corticoencephalic cell cultures subjected to metabolic damage and treatment with modulators of mitochondrial ATP-dependent potassium channels (mitoK(ATP)). In a first series of experiments, in vitro ischemic changes of the contents of purine and pyrimidine nucleoside diphosphates and triphosphates were measured by high performance liquid chromatography (HPLC) and the corresponding histological alterations were determined by celestine blue/acid fuchsin staining. As an ischemic stimulus, incubation with a glucose-free medium saturated with argon was used. Ischemia decreased the levels of adenosine, guanine and uridine triphosphate (ATP, GTP, UTP) and increased the levels of the respective dinucleotides ADP and UDP, whereas the GDP content was not changed. Both 5-hydroxydecanoate (5-HD) and diazoxide failed to alter the contents of nucleoside diphosphates and triphosphates, when applied under normoxic conditions. 5-HD (30 microM) prevented the ischemia-induced changes of nucleotide and nucleoside levels. Diazoxide (300 microM), either alone or in combination with 5-hydroxydecanoate (30 microM) was ineffective. Pyruvate (5 mM) partially reversed the effects of ischemia or ischemia plus 2-deoxyglucose (20mM) in the incubation medium. Diazoxide (300 microM) and 5-HD (30 microM) had no effect in the presence of pyruvate (5mM) and 2-deoxyglucose (20mM). Staining the cells with celestine blue/acid fuchsin in order to classify them as intact, reversibly or profoundly injured, revealed a protective effect of 5-HD. When compared with 5-HD, diazoxide, pyruvate and 2-deoxyglucose had similar but less pronounced effects. In conclusion, these results suggest a protective role of 5-hydroxydecanoate on early corticoencephalic nucleotide and cell viability alterations during ischemia.     

Characterization of the 31P NMR spectrum of the schistosome vector Biomphalaria glabrata and of the changes following infection by Schistosoma mansoni

The 31P nuclear magnetic resonance (NMR) spectrum of the digestive gland-gonad complex (DGG) of the schistosome vector Biomphalaria glabrata was characterized and the effects of infection by Schistosoma mansoni noted. The in vivo spectrum was comprised of 11 peaks, 5 downfield and 6 upfield of an external 85% phosphoric acid standard. Based on a variety of analytical procedures, the upfield peaks from the standard were demonstrated to be composed of carbamoyl phosphate + a mixture of 3 phosphatides and sphingomyelin, the gamma + beta phosphorus resonances of nucleotide triphosphate (NTP) and nucleotide diphosphate (NDP), respectively, the alpha phosphorus resonances of NTP + NDP, NAD(H) + the phosphorus resonance of uridine phosphate from uridine diphosphoglucose (UDPG), the phosphorus resonance of glucose phosphate from UDPG and, last, the beta phosphorus resonance of NTP. The downfield peaks were assigned as glycerophosphoryl choline, intracellular inorganic phosphate (Pi), sugar phosphates + phosphoryl choline, aminoethyl phosphonate (AEP), and ceramide AEP. T1 values for the in vivo NMR components were determined by inversion recovery. Infection produced distinct alterations in the levels of nonnucleotide components of the in vivo 31P NMR spectrum and the spectra of tissue extracts. Specifically, the levels of phosphonate, phospholipids, and carbamoyl phosphate were markedly reduced, and the relative level of Pi was increased. The potential significance of these changes to the parasite-host relationship was discussed. In contrast, starvation resulted in a decreased level of phosphonate only. The pH of the intact DGG was estimated by titrating the inorganic phosphate component of tissue extracts. The mean pH was 6.9 for both control and infected material.     

Senescence and declining reproductive potential: Insight into molecular mechanisms through testicular metabolomics

Aging is associated with structural and functional changes in the organism that result in the declining of its functioning. Postponed parenthood has renewed the interest in age-related decline of testicular function and male fertility. Still, little is known about the molecular mechanisms associated with testicular senescence and related decline of fertility. Here we sought to elucidate the molecular basis of metabolic changes associated with testicular aging and reproductive potential using an NMR-based metabolomics approach. Testicular metabolic profiles of rats from 3 to 24?months-of-age were analysed. An age-associated decrease in most antioxidant metabolites, like betaine, creatine and glutathione was observed. Amino acid content changed as early as 6?months-of-age, with an increase in branched chain and aromatic amino acids, accompanied by decrease of nucleotide synthesis (IMP, CMP, ATP). Testicular content of phospholipid precursors (choline, ethanolamine, myo-inositol, glycerol) increased with advanced age and was accompanied by a decrease in the levels of their phosphorylated products, suggesting compromised spermatogenesis. This is the first metabolomics study of testicular tissue of aged rats and we were able to identify metabolites associated with reproductive maturity from the onset to senescence. Our results provide evidence for an influence of aging on global testicular metabolome, as early as 6?months-of-age, with a profound alteration of several key metabolic pathways associated with the male reproductive potential.     

Biphasic cytotoxic mechanism of extracellular ATP on U-937 human histiocytic leukemia cells: involvement of adenosine generation

Since extracellular ATP can exhibit cytotoxic activity in vivo and in vitro, its application has been proposed as an alternative anticancer therapy. In this study we investigated the mechanisms of ATP-induced cytotoxicity in a human leukemic cell line (U-937). ATP added as a single dose exceeding 50 microM was cytostatic or even cytotoxic for U-937 cells. Interestingly, growth inhibition by ATP (50-3500 microM) showed a biphasic dose response. Up to 800 microM, ATP was cytotoxic in a dose-dependent manner (EC(50) 90 microM). In a range between 800 and 2500 microM, cell count was markedly higher despite the higher ATP concentrations. The cytotoxic effect of ATP could be antagonized by addition of uridine as a pyrimidine source and, alternatively, by addition of the nucleoside transmembrane inhibitor dipyridamole. The apoptosis-inducing adenosine A(3) receptor was not involved in measurable quantities, since (1) adenosine did not lead to an elevation of intracellular calcium levels, and (2) an unselective A(1-3) antagonist (ULS-II-80) could not abrogate the cytotoxic effect. Experiments monitoring extracellular nucleotide metabolism confirmed the assumption that the long-term production and continuous uptake of adenosine, which is extracellularly generated by degradation of ATP, led to an intracellular nucleotide imbalance with pyrimidine starvation. The biphasic dose response to higher ATP concentrations could be explained by the rapid degradation of lower ATP concentrations (300 microM) to adenosine by serum-derived enzymes, whereas higher concentrations (900 microM) only produced small amounts of adenosine due to forward inhibition of AMP hydrolysis by prolonged high ADP levels. FACS analysis revealed that at lower adenosine concentrations (300 microM) a reversible G(1) phase arrest of the cell cycle was induced, whereas higher concentrations (1000 microM) triggered apoptosis. Considering ATP as a potential cytostatic drug, our data have important implications concerning metabolic interactions of administered nucleotides.     

Alterations in pyrimidine nucleotide metabolism as an early signal during the execution of programmed cell death in tobacco BY-2 cells

Changes in pyrimidine metabolism were investigated during programmed cell death (PCD) of tobacco BY-2 cells, induced by a simultaneous increase in the endogenous levels of nitric oxide (NO) and hydrogen peroxide. The de novo synthesis of pyrimidine nucleotides was estimated by following the metabolic fate of the (14)C-labelled orotic acid, whereas the rates of salvage and degradation pathways were studied by measuring the respective incorporation of (14)C-labelled uridine and uracil under different treatments. Nucleic acid metabolism was also examined using labelled thymidine as a marker. The results show that specific alterations in the balance of pyrimidine nucleotide synthesis, which include a decreased rate of salvage activity of uracil and uridine and increased salvage activity of thymidine, represent a metabolic switch that establishes proper cellular conditions for the induction of PCD. In particular, a reduction in the utilization of uracil for salvage products occurs very early during PCD, before the appearance of typical cytological features of the death programme, thus representing an early metabolic marker for PCD. These changes are strictly associated with PCD, since they do not occur if NO or hydrogen peroxide are increased individually, or if actinomycin, which inhibits the death programme, is added into the medium in the presence of NO and hydrogen peroxide. The possible roles of these fluctuations in pyrimidine metabolism on the cellular nucleotide pool are discussed in relation to the induction of cell death.