ascorbic-acid and Pheochromocytoma

High levels of catecholamines in pheochromocytoma (PHEO) are associated with risk of cardiovascular complications. In this study, we looked for potential differences in markers of oxidative stress - vitamin C, superoxide dismutase (SOD) and malondialdehyde (MDA) in PHEO before and after the operation. We studied 18 subjects with PHEO who were examined before and approximately one year after the successful tumor removal (free of disease). All subjects had elevated urinary epinephrine and/or norepinephrine levels before the operation. Vitamin C levels increased significantly after the operation from 61+/-27 to 77+/-20 micromol/l (P=0.02), and MDA decreased significantly after the tumor removal from 2.6+/-0.4 to 2.0+/-0.6 micromol/l (P=0.01). However, no changes were found in SOD activity before and after the operation. In conclusion, increased catecholamine production in PHEO is accompanied by decreased levels of vitamin C and increased levels of MDA which may indicate the activation of oxidative stress in PHEO. Successful operation was associated with lowering of oxidative stress by using both biomarkers. On the contrary, no changes in SOD activity before and after the tumor removal were noted.

Topics: Ascorbic Acid; Biomarkers; Catecholamines; Female; Humans; Male; Malondialdehyde; Middle Aged; Oxidative Stress; Pheochromocytoma; Reactive Oxygen Species; Superoxide Dismutase; Treatment Outcome

Pheochromocytomas and paragangliomas (PCPG) are usually benign neuroendocrine tumors. However, PCPGs with mutations in the succinate dehydrogenase B subunit (. We studied the genetic alterations of cluster I PCPGs compared with cluster II PCPGs, which usually present as benign tumors. By targeting the signature molecular pathway, we investigated the therapeutic effect of ascorbic acid on PCPGs using. By investigating PCPG cells with low SDHB levels, we show that pseudohypoxia resulted in elevated expression of iron transport proteins, including transferrin (TF), transferrin receptor 2 (TFR2), and the divalent metal transporter 1 (. The data here demonstrate that targeting redox homeostasis as a cancer vulnerability with pharmacologic ascorbic acid is a promising therapeutic strategy for

Topics: Animals; Antioxidants; Apoptosis; Ascorbic Acid; Cell Line, Tumor; Disease Models, Animal; DNA Damage; Drug Screening Assays, Antitumor; Female; Gene Knockdown Techniques; Humans; Iron; Mice; Mutation; Oxidative Stress; Paraganglioma; Pheochromocytoma; Reactive Oxygen Species; Succinate Dehydrogenase

The guanine base is prone to oxidation by free radicals regardless of the cellular moiety it is bound to. However, under conditions of oxidative stress, 8-oxoguanosine triphosphate (oxo(8)GTP) formation has been shown to occur without oxidation of the guanine base in DNA. In vitro studies have suggested that oxo(8)GTP could impact G-protein signaling and RNA synthesis. Whether increased levels of oxo(8)GTP translate into cellular malfunction is unknown. Data presented herein show that oxo(8)GTP is formed in cell-free preparations as well as in PC12 cells after exposure to physiologically relevant oxidative conditions generated with 10 microM copper sulfate and 1 mM L-ascorbic acid (Cu/Asc). We also determined that oxo(8)GTP has biological activity as a potent inhibitor of nitric oxide-stimulated soluble guanylyl cyclase (sGC). The increase in oxo(8)GTP formation in purified GTP and PC12 cells exposed to Cu/Asc caused a significant reduction in the product of sGC activity, cGMP. This oxidation of GTP was attenuated by the addition of reduced glutathione under these same Cu/Asc conditions, thus preventing the decrease in sGC activity. This suggests that oxo(8)GTP is produced by free radicals in vivo and could have significant impact on cell functions regulated by sGC activity such as synaptic plasticity in the central nervous system.

Topics: Animals; Ascorbic Acid; Cell Extracts; Cell Line; Central Nervous System; Chromatography, High Pressure Liquid; Copper Sulfate; Cyclic GMP; Enzyme Activation; Free Radicals; Guanosine Triphosphate; Guanylate Cyclase; In Vitro Techniques; Nitric Oxide; Oxidation-Reduction; Oxidative Stress; Pheochromocytoma; Rats

In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 1.0 mm) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the l-type Ca2+ channel inhibitor nifedipine. The NO-donor (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3, 1.0 mm) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mm) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K+ (75 mm) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mm) with NOR-3 + high K+ restored high K+ effects. Co-infusion of nifedipine inhibited high K+-induced DA + 3-MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+-dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine-insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated.

Topics: 3,4-Dihydroxyphenylacetic Acid; Animals; Ascorbic Acid; Calcium; Calcium Channel Blockers; Cell Survival; Cyclic GMP; Dopamine; Enzyme Inhibitors; Extracellular Space; Homovanillic Acid; Microdialysis; Nitric Oxide; Nitric Oxide Donors; Nitro Compounds; PC12 Cells; Pheochromocytoma; Potassium; Rats; S-Nitroso-N-Acetylpenicillamine

By monitoring dopamine metabolism in rat pheochromocytoma derived PC12 cell cultures during extended treatment with manganese chloride, we assessed the functional changes occurring in a dopaminergic system during the development of manganese-induced damage. Besides eliciting a specific toxic effect on PC12 cells, manganese induced the complete disappearance of extracellular (free) but not intracellular (vesicle stored) dopamine and its metabolite 3,4-dihydroxyphenylacetic acid. This effect was observed also using low manganese concentrations (1 microM) and mainly occurred by non-enzymatic catechol oxidation since it was still evident in a cell free medium and it was fully prevented by ascorbic acid but not by reduced glutathione. The possibility of a mere "non-biological" action was ruled out by the observation of an irreversible effect of manganese as manifested by the cells' apparent inability to release dopamine or 3,4-dihydroxyphenylacetic acid into the culture medium even after complete manganese removal (post-manganese incubation). That a free radical mechanism was not involved in the genesis of this irreversible effect was shown by the fact that neither ascorbic acid, catalase, superoxide dismutase nor glutathione-peroxidase were able to prevent the decrease in catecholamine levels in the "post-manganese" incubation medium when added at the same time as the manganese. The results establish this PC12 cell system as an in vitro model for studying interactions between manganese and catechols and provide a detailed description of the nature of the neurochemical alterations that this heavy metal can induce in a dopaminergic system.

Topics: Adrenal Gland Neoplasms; Animals; Ascorbic Acid; Cell Survival; Chlorides; Culture Media; Dopamine; Dose-Response Relationship, Drug; L-Lactate Dehydrogenase; Manganese Compounds; Manganese Poisoning; Oxidation-Reduction; Pheochromocytoma; Tumor Cells, Cultured; Zinc

Tyrosine hydroxylase activity is reversibly modulated by the actions of a number of protein kinases and phosphoprotein phosphatases. A previous report from this laboratory showed that low-molecular-weight substances present in striatal extracts lead to an irreversible loss of tyrosine hydroxylase activity under cyclic AMP-dependent phosphorylation conditions. We report here that ascorbate is one agent that inactivates striatal tyrosine hydroxylase activity with an EC50 of 5.9 microM under phosphorylating conditions. Much higher concentrations (100 mM) fail to inactivate the enzyme under nonphosphorylating conditions. Isoascorbate (EC50, 11 microM) and dehydroascorbate (EC50, 970 microM) also inactivated tyrosine hydroxylase under phosphorylating but not under nonphosphorylating conditions. In contrast, ascorbate sulfate was inactive under phosphorylating conditions at concentrations up to 100 mM. Since the reduced compounds generate several reactive species in the presence of oxygen, the possible protecting effects of catalase, peroxidase, and superoxide dismutase were examined. None of these three enzymes, however, afforded any protection against inactivation. We also examined the effects of ascorbate and its congeners on the activity of tyrosine hydroxylase purified to near homogeneity from a rat pheochromocytoma. This purified enzyme was also inactivated by the same agents that inactivated the impure corpus striatal enzyme. Under conditions in which ascorbate almost completely abolished enzyme activity, we found no indication for significant proteolysis of the purified enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We also found that pretreatment of PC12 cells in culture for 4 h with 1 mM ascorbate, dehydroascorbate, or isoascorbate (but not ascorbate sulfate) also decreased tyrosine hydroxylase activity 25-50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adrenal Gland Neoplasms; Animals; Ascorbic Acid; Catalase; Chelating Agents; Colforsin; Corpus Striatum; Cyclic AMP; Glutathione; Male; Peroxidase; Pheochromocytoma; Phosphorylation; Protein Kinases; Rats; Rats, Inbred Strains; Sulfhydryl Compounds; Superoxide Dismutase; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

Topics: Ascorbic Acid; Bucladesine; Catecholamines; Cell Line; Kinetics; Nerve Growth Factors; Pheochromocytoma; Potassium

A clonal line of rat pheochromocytoma cells was used as a model of noradrenergic tissue to study ascorbic acid transport. These cells were used because, like sympathetic neurons, they synthesize large amounts of noradrenaline in the presence of ascorbate, they respond to nerve growth factor with the production of neurites and they release, store and take up catecholamines. In these cells, both with and without nerve growth factor (NGF) treatment, [14C]ascorbic acid was concentrated by a stereospecific saturable, energy dependent transport system that could be described by a Michaelis-Menten transport model. The Kt and Vmax for ascorbic acid were approximately 0.03 mM and 0.3 nmole per min per mg protein respectively for both untreated and NGF-treated cells. The ability of the cells to concentrate ascorbic acid was not due to intracellular binding. Cells untreated with NGF and loaded with [14C]ascorbic acid to a concentration of 5.6 nmoles per mg protein retained only 6% of the initial intracellular [14C]ascorbic acid after the 24 h in normal growth medium. Thus, although pheochromocytoma cells contain an ascorbate concentrating system, optimal production of noradrenaline requires ascorbate in the medium.

Topics: Adrenal Gland Neoplasms; Animals; Ascorbic Acid; Cells, Cultured; Kinetics; Nerve Growth Factors; Pheochromocytoma; Rats

A method is described for the estimation of adrenaline and noradrenaline in a 5 to 10 ml plasma sample.A batch technique of adsorption and elution from alumina is used and the final estimation is carried out using an automated fluorimetric technique based on the trihydroxyindole method. Fluorescence is stabilized first by thioglycollic acid to give noradrenaline concentrations only and then by ascorbic acid to give the total catecholamine concentration. The results of a number of catecholamine analyses are given.

Topics: Adsorption; Adult; Aluminum; Ascorbic Acid; Autoanalysis; Epinephrine; Fluorometry; Humans; Male; Methods; Norepinephrine; Pheochromocytoma; Thioglycolates; Urinary Bladder Neoplasms