Leonid Grinberg

Protective Effects of Tea Polyphenols against Oxidative Damage to Red Blood Cells

Tea polyphenols (TPP) from black and green teas were evaluated for their antioxidant effects on normal red blood cells (RBC) and beta-thalassemic RBC membranes challenged with exogenous oxidants in vitro. The TPP of both types protected RBC against primaquine-induced lysis; they also protected the whole cells and the membranes against H2O2-induced lipid peroxidation so that about 80% protection was reached at [TPP] = 10 microg/mL. TPP from black tea at the same concentration protected normal RBC from morphological alterations caused by the peroxide treatment. The mechanism of the effects of TPP was investigated using a chemical system generating .OH (iron + ascorbic acid). TPP from both black and green teas inhibited the .OH fluxes in a concentration-dependent manner, indicating the possibility of iron chelation by TPP. Spectrophotometric titration revealed that TPP could stoichiometrically bind ferric iron to form a redox-inactive Fe-TPP complex. Quantitative analysis suggests that one or more major catechins from the TPP preparations are the likely iron-binding compounds accounting for the antioxidant effects of TPP on RBC.

A low molecular weight copper chelator crosses the blood-brain barrier and attenuates experimental autoimmune encephalomyelitis

Increasing evidence suggests that enhanced production of reactive oxygen species (ROS) activates the MAP kinases, c‐Jun N‐terminal protein kinase (JNK) and mitogen‐activated protein kinase MAPK (p38). These phosphorylated intermediates at the stress‐activated pathway induce expression of matrix metalloproteinases (MMPs), leading to inflammatory responses and pathological damages involved in the etiology of multiple sclerosis (MS). Here we report that N‐acetylcysteine amide (AD4) crosses the blood–brain barrier (BBB), chelates Cu2+, which catalyzes free radical formation, and prevents ROS‐induced activation of JNK, p38 and MMP‐9. In the myelin oligodendrocyte glycoprotein (MOG)‐induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, oral administration of AD4 drastically reduced the clinical signs, inflammation, MMP‐9 activity, and protected axons from demylination damages. In agreement with the in vitro studies, we propose that ROS scavenging by AD4 in MOG‐treated animals prevented MMPs induction and subsequent damages through inhibition of MAPK pathway. The low toxicity of AD4 coupled with BBB penetration makes this compound an excellent potential candidate for the therapy of MS and other neurodegenerative disorders.

Protective effects of rutin against hemoglobin oxidation

A prooxidant drug, primaquine (PQ) was used to produce oxidative stress in human red blood cells (RBC) in vitro. Rutin, a plant flavonoid, did not prevent PQ-induced cell lysis but protected against hemoglobin (Hb) oxidation inside RBC. After PQ removal, rutin failed to reduce preformed met-Hb indicating that the rutin protective effect manifests only in the presence of PQ. Since H2O2 was proved to mediate PQ-induced Hb oxidation, authentic Hb was studied for its reaction with H2O2 and rutin in solution. Rutin partially protected oxy-Hb against H2O2-induced oxidation and heme loss. Rutin was also shown to delay H2O2-induced met-Hb oxidation to ferryl-Hb. Rutin directly reduced ferryl-Hb to met-Hb in stoichiometric (1:1) reaction characterized by a rate constant of 100 to 130/M/sec. It is assumed that by reducing ferryl-Hb, rutin prevents oxy-Hb from reacting with ferryl-Hb (comproportionation reaction), thus preventing half of the oxy-Hb molecules from being converted to met-Hb. This mechanism is consistent with 50% inhibition by rutin (at the maximum of its activity) of PQ-induced oxy-Hb oxidation in RBC. The present results demonstrate new antioxidant properties of rutin that may be useful in diminishing oxidative damage to pathological red blood cells.

Hydroxyl radical generation in β-thalassemic red blood cells☆

Abstract To provide more experimental evidence for the proposed role of oxygen free radicals in red blood cell (RBC) damage in β-thalassemia, hydroxyl radical generation was studied in thalassemic (Th) vs. normal (N) RBC. ḃ OH fluxes were quantified by the conversion of salicylic acid (SA) into its hydroxylated products, 2,3- and 2,5-dihydroxybenzoic acids (DHBA) and catechol, assayed with HPLC coupled to electrochemical detection. No significant difference in spontaneous ḃ OH generation between N-RBC and Th-RBC was found. Ascorbic acid (0.5–3.0 mM) induced many-fold increases in SA hydroxylation in a dose-dependant manner in both types of cells. In the presence of ascorbate (1.0 mM), the SA hydroxylated products were determined in Th-RBC vs. N-RBC as follows (nmol/ml): 2,5-DHBA, 1.45 ± 0.06 vs. 1.81 ± 0.05 ( p = 0.001); 2,3-DHBA, 1.89 ± 0.21 vs. 1.15 ± 0.08 ( p = 0.008) and catechol, 0.87 ± 0.13 vs. 0.38 ± 0.05 ( p = 0.006). The results showed significant increase in the total SA hydroxylation in Th-RBC as compared to N-RBC with a tendency to form 2,3-DHBA and catechol at the expanse of 2,5-DHBA. The excessive · OH generation in Th-RBC is attributed to the abnormally high content of redox active iron in the cytosolic and/or membrane compartments of these cells.

Studies on curcumin and curcuminoids: XXVI. Antioxidant effects of curcumin on the red blood cell membrane

Abstract A natural polyphenol curcumin (Cur) was tested for its antioxidant effects on human red blood cell (RBC) and their membranes. Cur, at concentrations of 4–100 μM protected RBC against H2O2-induced lysis and lipid peroxidation (LPO). Cur (100 μM) caused significant inhibition of the LPO in normal RBC ghosts supplemented with exogenous iron and in β-thalassemic (Th) ghosts containing endogenous iron deposits. The Cur protective effect on Th ghosts was much more pronounced when ascorbic acid was added in the system (prior to H2O2) aiming to induce partial iron reduction. Cur alone did not change the ratio of Fe2+ /Fe3+ in these ghosts but significantly inhibited the rate of Fe2+ reoxidation by H2O2. The results indicate the possibility of Fe2+ complexing with Cur leading to a lower iron reactivity towards H2O2. Therefore Cur as a membrane antioxidant may protect Th RBC against iron-catalyzed oxidative damage.

Nitroxide stable radical prevents primaquine-induced lysis of red blood cells

Primaquine (PQ), an antimalarial drug, is known to produce multiple oxidative effects in red blood cells (RBC). Because H2O2, OH and intracellular superoxide are implicated in this oxidation, the effect of cell-permeable nitroxide 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) capable of scavenging O2.- has been studied. PQ caused RBC lysis and facilitated the oxidation of oxyhemoglobin (oxyHb) to methemoglobin (MetHb). The lysis was partially inhibited by catalase and by the metal chelating agent 2,2-dipyridyl. TEMPO blocked PQ-induced RBC lysis in dose-dependent manner (2 mM IC50) but enhanced the oxidation of oxyHb to MetHb. PQ facilitated the lysis also in the presence of CO but without effecting Hb oxidation. This hemolysis, however, was inhibited by TEMPO. The results indicated that: (a) no causative relationship exists between PQ-induced Hb oxidation and RBC lysis; (b) TEMPO can directly oxidize heme-iron without causing membrane injury; (c) the aerobic toxicity of PQ in this system is mediated by O2.- and H2O2 and possibly by redox-active labile metals (d) TEMPO can protect by detoxifying O2.- and oxidizing reduced labile metal ions and thus blocking their participation in Fenton reaction.

Methionine-centered redox cycle in organs of the aero-digestive tract of young and old rats.

It is commonly accepted that aging is associated with a decline in the antioxidant defense of the cell; accordingly, certain redox enzymes are used as markers of biological senescence. To further test and specify this general concept, we studied age-related changes in the enzymes of the methionine-centered redox cycle (MCRC) in four aero-digestive organs of rats. The levels of cytosolic thioredoxin (Trx), thioredoxin reductase (TrxR), and methionine sulfoxide reductase (Msr), all tended to decline with age. The enzymatic activities of MsrA and MsrB were significantly lower in the organs of aged animals. In general, the magnitude of this decline increased in the order: tongue < sternohyoid muscle < larynx < esophagus. The relative stability of MCRC in the old tongues might be part of the well-preserved oxidative metabolism as confirmed by the age-related increase in mitochondrial marker and muscle tissue in these tongues. In total, the results suggest that age-associated oxidative damage is organ-specific and could reflect differences in morphological composition of these tissues, and among them, relative content of striated muscles.

Primaquine-induced superoxde production by β-thalassemic red blood cells

Abstract Primaquine, a prooxidant antimalarial drug, incubated with human red blood cells (RBC) induced marked superoxide generation in the cells as detected by exogenous cytochrome c reduction. In the presence of primaquine, β-thalassemic RBC produced significantly more superoxide than normal RBC, thus reflecting the vulnerability of β-thalassemic cells to oxidative stress.

Iron, ferritin and proteins of the methionine-centered redox cycle in young and old rat hearts.

Progressive oxidation of cellular components constitutes a major mechanism of the aging process. An emerging paradigm of redox signaling suggests that low level oxidants activate protective pathways resulting in prolonged cell survival. This report centers on the study of cardiac muscle in young and old rats, including (i) the expression of ferritin (Ft) the major iron storage protein, and (ii) the expression of the major proteins of the methionine-centered redox cycle (MCRC), which controls the cellular methionine redox status. Total amounts of Ft (protein) and its mRNA encoding for Ft L-subunit (Ft-L) were higher in the aged hearts, indicating that the iron-binding capacity of myocardial Ft increased with age. Among the proteins of the MCRC, methionine sulfoxide reductases A and B (MsrA, MsrB) and MsrA mRNA were significantly higher in hearts of old rats with a significant decrease in MsrA activity. The observed up-regulation of the expression of Msr and Ft-L could represent a protective response to the increased oxidative stress in the aging myocardium.

Photoreactivity of biologically active compounds: XI. Primaquine and metabolites as radical inducers

Abstract Reduction of ferricytochrome C and oxidation of haemoglobin was used to examine redox properties of primaquine, metabolites and photodegradation products of the drug. The influence of oxygen radicals (O 2 ·− and OH·) were studied by the addition of oxygen radical scavengers. Photodecomposition of primaquine (80 mW/cm 2 , xenon lamp, 290–800 nm) prior to dark-incubation resulted in a substantial accelerated drug-induced O 2 ·− formation and haemoglobin oxidation. Formation of OH· (dark reaction) could be detected after photochemical degradation of primaquine. In the presence of erythrocytes the formation of oxygen radicals induced by the photodecomposition products was even more pronounced. A high oxygen content in the medium during irradiation accelerated the photodecomposition-rate of primaquine. The metabolite 6-desmethyl primaquine was a more potent O 2 ·− producer and haemoglobin oxidizer than primaquine (dark reactions). During irradiation (80 mW/cm 2 , 290–800 nm) primaquine formed more O 2 ⋅− and produced a detectable level of OH· compared to the dark reactions.