Impact of small fractions of abnormal erythrocytes on blood rheology.

Red blood cell (RBC) populations are inherently heterogeneous, given mature RBC lack the transcriptional machinery to re-synthesize proteins affected during in vivo aging. Clearance of older, less functional cells thus aids in maintaining consistent hemorheological properties. Scenarios occur, however, where portions of mechanically impaired RBC are re-introduced into blood (e.g., damaged from circulatory support, blood transfusion) and may alter whole blood fluid behavior. Given such perturbations are associated with poor clinical outcomes, determining the tolerable level of abnormal RBC in blood is valuable. Thus, the current study aimed to define the critical threshold of blood fluid properties to re-infused physically-impaired RBC. Cell mechanics of RBC were impaired through membrane cross-linking (glutaraldehyde) or intracellular oxidation (phenazine methosulfate). Mechanically impaired RBC were progressively re-introduced into the native cell population. Negative alterations of cellular deformability and high shear blood viscosity were observed following additions of only 1-5% rigidified RBC. Low-shear blood viscosity was conversely decreased following addition of glutaraldehyde-treated cells; high-resolution microscopy of these mixed cell populations revealed decreased capacity to form reversible aggregates and decreased aggregate size. Mixed RBC populations, when exposed to supraphysiological shear, presented with compounded mechanical impairment. Collectively, key determinants of blood flow behavior are sensitive to mechanical perturbations in RBC, even when only 1-5% of the cell population is affected. Given this fraction is well-below the volume of rigidified RBC introduced during circulatory support or transfusion practice, it is plausible that some adverse events following surgery and/or transfusion may be related to impaired blood fluidity.

Quercetin suppresses intracellular ROS formation, MMP activation, and cell motility in human fibrosarcoma cells.

Cell metastasis is a major cause of death from cancer and can arise from excessive levels of oxidative stress. The objective of this study was to investigate whether the natural flavonoid quercetin can inhibit matrix metalloproteinase (MMP)-2 and -9 activities through the attenuation of reactive oxygen species (ROS) formation, an event expected to lead to the inhibition of cell motility. To induce sustained ROS formation, cells were treated with phenazine methosulfate (PMS; 1 µM). Noncytotoxic concentrations of quercetin inhibited PMS-induced increases in cell motility in HT1080 human fibrosarcoma (HT1080) cells. While nearly 100% of cells were observed to migrate after 24 h of PMS treatment, quercetin significantly (P < 0.01) suppressed this effect. We also found that quercetin, up to 10 µg/mL, attenuated PMS-induced MMP-2 activation. We then investigated whether the decreased levels of MMP-2 activation could be attributable to lower levels of ROS formation by quercetin. We found that quercetin treatments significantly attenuated PMS-induced ROS formation (P < 0.01) and resulted in decreased cell motility associated with a reduction in MMP-2 and -9 activitiy in HT1080 cells, even in the absence of PMS treatment. Collectively, these results suggest that quercetin inhibits cell motility via the inhibition of MMP activation in HT1080 cells in the presence and absence of PMS. This is likely to be a result of the suppression of intracellular ROS formation by quercetin.

In vitro effect of calcium dobesilate on oxidative/inflammatory stress in human varicose veins.

AIM: To determine whether calcium dobesilate can act in chronic venous insufficiency by similar antioxidant, anti-inflammatory mechanisms as in diabetic retinopathy. METHODS: Calcium dobesilate was tested in vitro for its protective action against oxidative/inflammatory stress in human varicose veins. Varicose greater saphenous veins were obtained from 14 patients (11 men, 3 women) aged 53-65 years. Oxidative stress was induced exogenously in the vein segments, with the phenazine methosulphate (PMS)/NADH couple. Total antioxidant status (TAS) and malondialdehyde (MDA) contents were used as markers of oxidative stress. RESULTS: Calcium dobesilate significantly prevented oxidative disturbances in the micromolar range. PMS/NADH-dependent TAS decrease was fully prevented with IC(50) = 11.4 ± 2.3 µmol/L (n = 6 veins), whereas MDA increase was fully prevented with IC(50) = (102 ± -3) µmol/L (n = 6 veins). Calcium dobesilate acted quali- and quantitatively like rutin, the reference compound. Comparison with pharmacokinetic data suggests that calcium dobesilate can act at therapeutic concentrations. CONCLUSION: Calcium dobesilate protected human varicose veins against oxidative stress in vitro at levels that correspond to therapeutic concentrations. Further studies are required to investigate whether a similar action is found in varicose veins from patients orally treated with calcium dobesilate.

The action of phenazine methosulphate in causing cellular damage in the isolated mouse soleus muscle preparation.

Exposure of the isolated mouse soleus preparation to 1.0 mM phenazine methosulphate (PMS) caused (i) a slow and modest release of creatine kinase (CK) that was exacerbated by removal of extracellular Ca2+, (ii) a specific type of ultrastructural damage, namely a characteristic spacing of the myofibrils, (iii) swelling of the mitochondria, indicating a modest rise in [Ca2+]i, and (iv) swelling of the sarcoplasmic reticulum (SR). It is suggested that PMS (i) activates a sarcolemma oxidoreductase which synergistically interacts with raised [Ca2+]i to cause modest CK efflux and (ii) activates an oxidoreductase on the SR, thereby generating electrons which directly modify the integrity and organisation of the contractile apparatus.

Measurement of gamma-enolase release, a new method for selective quantification of neurotoxicity independently from glial lysis.

We have developed a sensitive enzymatic-immunoassay to quantify the level of gamma-enolase (a specific neuronal enzyme) which is released from cultured cells after exposure to various toxins. We show that this method can estimate selectively neuronal cell death without significantly interfering with glial cell death. Indeed, no gamma-enolase is released when glial cells are killed with free-radical producing agents. Experiments comparing the levels of neuronal cell death induced by NMDA or free-radical producing drugs, performed either by measuring gamma-enolase release or using the classical fluorescein diacetate method, yielded similar results. In addition to selectively follow neuronal death in a mixed population of neurons and glial cells, this method provides a way of determining the cell death kinetics from a single culture dish, since enolase can be measured on small samples taken from the culture medium. Finally, we propose these two methods as being complementary and useful neuronal and other cellular death indexes and also to understand the complex problem of glial influence on neuronal survival or death.

Movement of methylphenazyl free radicals in polar and nonpolar liquids.

A protonated, charged free radical of methylphenazine methosulfate (PMS) was generated at carbon electrodes in a buffered aqueous medium. This radical diffused from the aqueous phase into nonpolar organic solvents, where it was stable for extended periods. The electron spin resonance (ESR) spectrum of the free radical species in the nonpolar solvent was significantly different from that of the aqueous species. This difference was attributed to the loss of electric charge through deprotonation at the solution interface, followed by solvation of the uncharged species in the organic phase. ESR spectra are presented for PMS free radicals in polar and nonpolar liquid phases, along with electrochemical results and conclusions regarding the mechanisms of movement and toxicity of phenazyl free radicals in biological systems.

Sarcolemma blebs and cell damage in mammalian skeletal muscle.

Plasma membrane blebs are an early sign of cellular damage in isolated cells. Phenazine methosulphate (PMS) triggers the production of conspicuous and characteristic sarcolemma blebs in mouse diaphragm skeletal muscle incubated in vitro and also causes severe myofilament damage. It is suggested that PMS activates transmembrane NAD(P)H dehydrogenases and, in turn, a modification of sulphydryl groups of the cytoskeleton, thereby permitting bleb formation in contracting cells.

Cytotoxicity of phenazine methosulphate on skeletal muscle. The role of the sarcoplasmic reticulum in initiating myofilament damage.

Phenazine methosulphate (PMS) or ferricyanide caused ultrastructural damage, including sarcolemma folds and swelling of the sarcoplasmic reticulum (SR), in amphibian skeletal muscle which corresponds with that triggered by a rise in [Ca]i and which, it is suggested, is caused by the activation of NAD(P)H oxidases at the sarcolemma (where it causes sarcolemma folding) and SR (where it causes myofilament damage). PMS also caused SR swelling and more limited damage in chemically-skinned muscle at zero [Ca]. In contrast with the oxygen paradox of cardiac muscle, there is no evidence for the production of oxygen radicals since no protection was provided by N2, mannitol, desferrioxamine or alpha-tocopherol, nor was the cell damage produced by an influx of Ca across the sarcolemma.

Cytotoxicity of phenazine methosulfate in isolated rat hepatocytes is associated with superoxide anion production, thiol oxidation and alterations in intracellular calcium ion homeostasis.

The metabolism of phenazine methosulfate (PMS) by isolated rat hepatocytes is associated with superoxide anion production, and with a substantial decrease in intracellular levels of reduced glutathione, most of which is oxidized to GSSG. A marked loss of protein-free sulfhydryl groups also occurs when intracellular glutathione is depleted, and cytotoxicity follows. These effects are associated with the inhibition of the plasma membrane Ca2+-ATPase and with intracellular accumulation of calcium ion which is preferentially sequestered in mitochondria. Maintenance of protein sulfhydryl groups in the reduced state by dithiothreitol (DTT) prevents the alterations in intracellular calcium homeostasis and protects against toxicity.

Study on the "toxic oxygen effect" of Janus green B in mouse ascites tumour cells.

10(7) mouse ascites tumor cells/ml incubated at 37 degrees C in 0.5 to 1.0 X 10(-4) M Janus green B or in 1.0 X 10(-4) M phenazine methosulphate are destroyed in 100 per cent oxygen atmosphere but remain transplantable in nitrogen atmosphere. The "sensitizing" effect of oxygen can be substituted by SH inhibitors (iodoacetic acid, iodoacetamide and their spinlabelled variants) as well as by some nitroxide free radicals. The "oxygen effect" is blocked by mercaptoethanole or cooling. Compared with the spectrum of native cells a more symmetrical singlet of larger amplitude, approximately g = 2 value, arose in the ESR spectrum of Janus green B treated cells. The "oxygen effect" observed in the presence of Janus green B differs in several ways from the oxygen effect of ionizing radiation and from the "photodynamic" effect.