Novel insights into the electrochemical detection of nitric oxide in biological systems.

In recent years, microsensor technologies have made a rapid expansion into different fields of physical sciences, engineering, and biomedicine. For analyses of various biomolecules, novel sensors and detection platforms in the electrochemical field have been reported recently. The most important applications based on microelectromechanical systems dramatically reduce the need of manipulation steps with samples, while improving data quality and quantitative capabilities. This is also the case of a special class of electrochemical sensors that allow direct, real-time and non-invasive measurements of nitric oxide, whose determination is crucial for the purposes of basic research, as well as of preclinical and clinical studies. Therefore, this minireview will focus on the description of recent discoveries in the electrochemical determination of nitric oxide, released in different in vitro systems.

The effects of chloroquine and hydroxychloroquine on nitric oxide production in RAW 264.7 and bone marrow-derived macrophages.

Chloroquine, an antimalarial drug, can also be used in the regulation of the immune system, e.g. it is used in the treatment of autoimmune diseases. In this study we investigated the effects of chloroquine and its hydroxy-derivative on nitric oxide (NO) production in two different cell types: (i) immortalized mouse macrophage cell line RAW 264.7 and (ii) mouse bone marrow-derived macrophages (BMDM). The cells were treated with different concentrations (1-100 µM) of chloroquine or hydroxychloroquine and stimulated with lipopolysaccharide for 24 h to induce NO production. Measurement of nitrites by the Griess reaction was used to evaluate the production of NO. Expression of inducible NO synthase was evaluated with Western blot and ATPcytotoxicity test was used to measure the viability of the cells. Our results showed that both chloroquine and its hydroxy-derivative inhibited NO production in both cell types. However, based on the results of LD50 these inhibitory effects of both derivatives were due to their cytotoxicity. The LD50 values for chloroquine were 24.77 µM (RAW 264.7) and 24.86 µM (BMDM), the LD50 for hydroxychloroquine were 13.28 µM (RAW 264.7) and 13.98 µM (BMDM). In conclusion, hydroxychloroquine was more cytotoxic than its parent molecule. Comparing the two cell types tested, our data suggest that there are no differences in cytotoxicity of chloroquine or hydroxychloroquine for primary cells (BMDM) or immortalized cell line (RAW 264.7).

The influence of wine polyphenols on reactive oxygen and nitrogen species production by murine macrophages RAW 264.7.

The aim was to study the antioxidant properties of four wine polyphenols (flavonoids catechin, epicatechin, and quercetin, and hydroxystilbene resveratrol). All three flavonoids exerted significant and dose-dependent scavenging effects against peroxyl radical and nitric oxide in chemical systems. The scavenging effect of resveratrol was significantly lower. All polyphenols decreased production of reactive oxygen species (ROS) by RAW264.7 macrophages. Only quercetin quenched ROS produced by lipopolysaccharide-stimulated RAW264.7 macrophages incubated for 24 h with polyphenols. Quercetin and resveratrol decreased the release of nitric oxide by these cells in a dose-dependent manner which corresponded to a decrease in iNOS expression in the case of quercetin. In conclusion, the higher number of hydroxyl substituents is an important structural feature of flavonoids in respect to their scavenging activity against ROS and nitric oxide, while C-2,3 double bond (present in quercetin and resveratrol) might be important for inhibition of ROS and nitric oxide production by RAW 264.7 macrophages.

Blood phagocyte activation during open heart surgery with cardiopulmonary bypass.

Open heart surgery with a cardiopulmonary bypass (CPB) is associated with a systemic inflammatory response which significantly contributes to adverse postoperative complications. The purpose of this study was to characterize the activation of blood phagocytes during open heart surgery with CPB. Blood samples were collected during and up to 24 h after surgery. The production of reactive oxygen species (ROS) in whole blood, the expression of surface molecules by blood phagocytes and complement activity in the plasma were determined. A cDNA microarray analysis of leukocyte RNA profile of genes was performed related to the inflammatory response. Activation of the complement was already observed at the beginning of CPB. This was followed by an increase in the neutrophil number and in both spontaneous and opsonized zymosan-activated ROS production after the onset of reperfusion. The activation of blood phagocytes was affirmed by changes in surface receptors involved in the adhesion and migration of leukocytes (CD11b, CD62L and CD31). Gene arrays also confirmed the activation of leukocytes 4 h after reperfusion. In conclusion, open heart surgery with a cardiopulmonary bypass was found to be associated with a rapid and pronounced activation of blood phagocytes and complement activation which was partly independent at the onset of CPB.

Total antioxidant capacity of serum increased in early but not late period after intestinal ischemia in rats.

The ischemia of small intestine was induced in anesthetized Wistar rats by occluding the superior mesenteric artery for 45 min and then the reperfusion was set. Serum samples were obtained at the end of the ischemic period and also during early (1 to 4 h) and late postischemic period (1 to 4 d). The total antioxidant capacity (TRAP) of serum samples was evaluated using luminol enhanced chemiluminescence. The increased mobilization of phagocytic cells and the release of reactive oxygen species into the circulation was observed from the first and second hour of the postischemic period, respectively. Nevertheless, the activity of natural antioxidant mechanisms of serum was already elicited at the end of the ischemic period. Furthermore, the TRAP of serum increased with the increasing duration of early postischemic period. Among the antioxidants studied, urate and ascorbate concentrations exerted the highest correlation with TRAP, but 31.6% of the total antioxidant capacity remained for the activity of an unidentified antioxidant(s). After being exhausted, the TRAP of serum oscillated around the preoperation level at days 1-4 of the postischemic period. The increase in total antioxidant capacity of serum induced by oxidative stress was sufficient to prevent lipoperoxidation both in serum and intestinal tissue.

Leukocyte mobilization, chemiluminescence response, and antioxidative capacity of the blood in intestinal ischemia and reperfusion.

Intestinal ischemia and reperfusion elicits changes in leukocyte counts and increased production of reactive oxygen species (ROS). The purpose of this study was to investigate whether these changes were followed by and/or connected with changes in the extracellular antioxidative capacity in a rat superior mesenteric artery (SMA) occlusion/reperfusion model. The SMA was occluded for 45 min and then allowed to be reperfused. Changes of leukocyte, polymorphonuclear (PMN), and lymphocyte counts, chemiluminescence (CL) of whole blood samples as a marker of ROS production, and the total antioxidative capacity of the serum were quantified at the end of ischemia and in 1 h intervals during the postischemic period up to 4 h. The myeloperoxidase (MPO) activity in the serum and intestinal tissue samples was also determined. The MPO activity in the intestinal tissue samples was significantly elevated at the end of ischemia, and this elevation lasted for the whole postischemic period. The oxidative challenge to the body induced a fast mobilization of extracellular antioxidative mechanisms already at the end of ischemia, which was followed by a significant increase in PMN counts and whole blood CL starting at the 2nd hour after reperfusion. The increased CL activity of whole blood was attributed to the increase of the circulating PMNs. No significant changes were observed in leukocyte and lymphocyte counts. It is concluded that compensatory mechanisms of the oxidative-antioxidative balance of the body react very quickly if challenged.

Effects of drugs inhibiting prostaglandin or leukotriene biosynthesis on postirradiation haematopoiesis in mouse.

Two non-steroidal anti-inflammatory drugs, i.e. indomethacin (INDO), an inhibitor of prostaglandin production, and esculetin (ESCUL), an inhibitor of leukotriene production, were tested for their ability to modify haematopoiesis in three experimental systems: (a) in vitro clonal proliferation of marrow GM-CFC from the irradiated mouse was found to be augmented by addition of INDO at a low concentration, and inhibited by ESCUL in a dose-dependent manner; (b) in the lethally irradiated and bone marrow-transplanted mice treated with the drugs in the postirradiation period, stimulatory effects of INDO on CFU-S and GM-CFC populations and an inhibitory effect of ESCUL on GM-CFC were observed; and (c) when the drugs were administered i.p. to mice 1 h before 5-Gy irradiation, INDO enhanced the postirradiation recovery of haematopoietic indices such the numbers of CFU-S, GM-CFC, peripheral blood granulocytes, and nucleated bone marrow cells, while ESCUL had no effect or even inhibited the recovery of these indices. Survival curves for CFU-S and GM-CFC showed that altered haematopoietic recovery in the INDO- and ESCUL-pretreated mice was not due to changes of intrinsic radiosensitivity of pluripotent (CFU-S) or committed (GM-CFC) stem cell populations. These results confirm earlier findings suggesting an inhibitory role of prostaglandins on haematopoiesis, and provide evidence that endogenous leukotrienes might play a positive role in the regulation of haematopoietic functions in an irradiated organism.

Cadmium ions influence the chemiluminescence activity of murine splenocytes both in vitro and in vivo.

The luminol-enhanced chemiluminescence (CL) activity of splenocytes of mice of the strain (CBA x C57B1)F1 was monitored after treatment with Cd2+ (cadmium chloride) in both in vitro and in vivo experiments. Cd2+ (at concentrations of 1 microM-1 mM) increased the CL reaction of the splenocytes (2 x 10(6) cells/ml) in vitro in a dose-dependent manner, both instantaneously and after incubation for 1 h. In in vivo experiments, Cd2+ was administered in two ways. Following a 14-day administration of cadmium to mice in drinking water (300 mg Cd2+/l), the CL reaction of the splenocytes was significantly reduced. On the other hand, after i.p. administration of CdCl2 dissolved in PBS (2 mg/kg body mass, repeated seven times during 14 days), the metabolic activity of the phagocytes was increased. From the results it follows that cadmium affects the immune system. However, its toxicity is dependent on the route of administration.

The effect of intestinal ischemia duration on changes in plasma antioxidant defense status in rats.

The purpose of this study was to follow up the changes in antioxidative adaptive mechanisms induced by various periods of small intestinal ischemia in Wistar rats. The superior mesenteric artery was occluded for 15, 30, 45, 60 and 90 min. After the respective ischemic intervals, a reperfusion was set for 120 min. Samples of the serum and intestinal mucosa were taken at the end of ischemia or at the end of reperfusion. Total radical-trapping antioxidant parameter (TRAP) of the serum and the oxidative burst of neutrophils were evaluated using luminol-enhanced chemiluminescence. Individual antioxidants in the serum and the concentration of thiobarbituric acid reactive substances (TBARs) in both serum and intestinal mucosa were measured spectrophotometrically. Increased activation of circulating neutrophils was found after the reperfusion irrespective of the duration of ischemia. TRAP of the serum was increased at the end of the ischemia lasting from 30 to 90 min. This effect was further enhanced by the subsequent reperfusion period. Ascorbate and urate contributed considerably to the TRAP value especially after reperfusion following 60 and 90 min of ischemia. On the other hand, no significant changes in albumin and bilirubin serum concentrations were observed. Contrary to the mobilized antioxidative mechanisms, increased lipid peroxidation was observed in both serum and mucosa samples.

IL-10 does not affect oxidative burst and expression of selected surface antigen on human blood phagocytes in vitro.

Cytokines play a major role in the control of inflammatory responses, participate in the regulation of blood phagocyte activities and as such are used for immunomodulatory therapy. In the present study, the influence of IL-10 on human blood phagocyte activity in the presence/absence of IL-6, IL-8 and TNF-alpha was tested in vitro. Our research analyzed the effects of cytokines on the production of reactive oxygen species measured by chemiluminescence and flow cytometry, and on the expression of surface molecules (CD11b, CD15, CD62L, CD31) measured by flow cytometry. IL-10 had no inhibitory effect on reactive oxygen species production and the expression of any examined adhesion molecule by resting or stimulated blood phagocytes within 3 h of incubation. Conversely, TNF-alpha, IL-6, and IL-8 increased reactive oxygen species production and the expression of CD11b and CD15 on both neutrophils and monocytes and decreased the expression of CD62L. These priming effects of the tested pro-inflammatory cytokines were not affected by IL-10. The obtained results suggest that IL-10 does not directly control blood phagocyte activation. These results also provide better information about the contribution of IL-6, IL-8 and TNF-alpha to the regulation of blood phagocyte-mediated inflammatory processes.

Human neutrophil mobilization during open heart surgery.

Phagocyte released reactive oxygen species are often discussed in connection with ischemic and reperfusion injuries to the myocardium. The kinetics of the accumulation and oxidative burst of human blood phagocytes was studied by chemiluminescence during open heart surgery in the myocardium of human patients. Direct evidence is presented for an accumulation of neutrophils along with their markedly increased metabolic activity (oxygen radical formation), especially following the reperfusion of the ischemic myocardium. Leukocyte numbers and activity remained significantly elevated even in the venous blood obtained 24 h after the operation.

Time course of leukocyte response and free radical release in an early reperfusion injury of the superior mesenteric artery.

The sequence of changes in circulating immune cells and in free radical production was studied during the small intestine reperfusion. Rat small intestine ischemia/reperfusion was induced by a 45 min superior mesenteric artery occlusion followed by a 4-hour reperfusion. Samples of peripheral blood were collected every 20 min during reperfusion. While the number of polymorphonuclear leukocytes increased significantly both in the sham-operated controls and the experimental group (about 400 per cent at the end of reperfusion), a decrease in lymphocyte counts to 60 per cent was observed in the experimental group only. Although there were no changes in the counts of total T lymphocytes, a significant reduction in B cell counts was observed. Flow-cytometrical measurements showed no changes in the Tc subpopulation, while the Th subpopulation increased in the experimental group only. Free radical generation in blood (luminometric measurements) increased gradually and reached an eight-fold level by the end of reperfusion in both groups. Thus, it has been shown that the increase in free radical production is mainly due to the increased number of polymorphonuclear leukocytes mobilized already at the initial stages of reperfusion. The reduction in B lymphocyte population is probably due to homing mechanisms

Phagocyte-induced oxidative stress in patients with haemodialysis treatment and organ transplantation.

The aim of the study was to examine the phagocyte-derived production of reactive oxygen species (ROS), lipoperoxidation and the total radical-trapping antioxidant parameter (TRAP) in patients undergoing regular haemodialysis (HD) followed by kidney transplantation (n = 13). A significant increase in both spontaneous and activated ROS generation was found during the post-transplantation period in days 1-3. This effect was caused mainly by an increased number of neutrophils. On the other hand, the TRAP parameter was decreased in HD patients. After kidney transplantation, the TRAP parameter increased, reaching the level of healthy controls at the end of the first week after surgery. Increase in MDA level was determined after HD and kidney transplantation. It can be concluded from the results obtained that phagocyte mobilisation and increased oxidative stress after HD and kidney transplantation were associated with the insufficient activity of extracellular antioxidant mechanisms resulting in increased lipid peroxidation.

Kinetics of luminol-enhanced chemiluminescence induced in murine splenocytes and bone marrow cells by various stimulating agents.

The luminol-amplified chemiluminescence (CL) activity of murine splenocytes and bone marrow cells was investigated and compared. Phagocytosis was activated by adding starch grains, opsonized zymosan particles (OZP), or phorbol myristate acetate (PMA) to cell suspensions. CL was studied within a concentration range of 3.13 x 10(6)-1.25 x 10(8) splenocytes/ml, and 1.56 x 10(6)-6.25 x 10(7) bone marrow cells/ml. The CL reaction intensity was increasing with rising cell concentration throughout the whole range studied. Starch grains and OZP elicited a unimodal kinetics of the CL response with a single peak after 10 min for splenocytes and 13-16 min for bone marrow cells. PMA activation induced a bimodal reaction with the first peak appearing after 3 min for either of the cell populations, and the second maximal peak being recorded after 6-7 min for splenocytes and 9-10 min for bone marrow cells.

The effect of microwave radiation on the primary IgM response to sheep red blood cells in mice.

We investigated the changes in the number of plaque-forming cells (PFC) in spleens of CBA mice after microwave (MW) irradiation. The mice were immunized with sheep red blood cells, the number of PFC was determined on days 4 and 5 after immunization. Mice received single doses of MW irradiation on days 1, 2, or 3 after immunization (exposure duration 1 to 9 min). It was found that the number of PFC was changed after MW irradiation and that this effect depended on the absorbed dose. Shorter exposures for 1, 3, and 5 min (SA = 4, 12, and 20.1 kJ/kg) stimulated the formation of PFC, exposures for 7 and 9 min did not change the number of PFC. After MW heating (exposure 5 min), the rectal temperature of mice was elevated by 2.5 degrees C. If the same thermal effect was induced by elevating the environmental temperature, the number of PFC was not increased. The observed changes in PFC number are probably due to the specific effect of MW radiation.

Effects of nordihydroguaiaretic acid on the chemiluminescence of murine phagocytes.

Antioxidative effects of nordihydroguaiaretic acid (NDGA) on murine bone marrow phagocytes were studied using luminol- and lucigenin-amplified chemiluminescence (CL). NDGA applied in vitro strongly suppressed the opsonized zymosan particles stimulated CL response in a dose-dependent manner (in concentrations of 0.3-30 micrograms NDGA/ml), thus confirming its antioxidant activity. However, no effects were observed in mice and their serum samples when investigated one minute to one hour after an i.p. administration of NDGA. These differences suggest that NDGA may undergo a rapid metabolism in vivo.

Influence of polysulfone and hemophan hemodialysis membranes on phagocytes.

The aim of the study was to compare the effect of hemophane and polysulfone membranes on the phagocyte-derived production of reactive oxygen species (ROS) as well as on neutrophil CD11b and CD62L expression in patients undergoing regular hemodialysis. The effects of hemodialysis membranes were also studied in in vitro conditions after coincubating them with differentiated HL-60 cells. ROS production was measured using chemiluminometric and flow cytometric methods. Expression of CD11b, CD62L and mitochondrial membrane potential were detected by monoclonal antibodies and by the JC-1 fluorescent probe, respectively. Depressed ROS production was observed in patients already before dialysis. Further decrease in ROS production and an increase in CD11b expression were observed especially in patients after hemophan hemodialysis. Decreased ROS production and increased CD11b expression were observed also after incubation of HL-60 cells with hemophan membranes. Mitochondrial membrane potential dropped only after incubating cells with hemophan membranes proving its more serious adverse effects in comparison with the polysulfone membrane. In conclusion, deleterious effects of hemodialysis on the metabolic activity of phagocytes were proved. Combining chemiluminescent and flow cytometric methods for the detection of ROS production and determining mitochondrial membrane potential can be useful tools for the analysis of material biocompatibility.

Comparison of the bioluminescence of Photorhabdus species and subspecies type strains.

Natural bioluminescence of all recently accepted Photorhabdus species and subspecies type strains (bacterial symbionts of entomopathogenic nematodes) was measured using a commercial luminometer; optimum conditions for the measurement were described. Cultures emitted reliably measurable bioluminescence with characteristic level and kinetics for each strain. Bioluminescence of all strains was significantly higher at 37 than at 25 degrees C at the beginning of the measurement, no effect of bacterial concentration on the intensity of bioluminescence was observed. The technique can provide reliable and quick information for the determination of Photorhabdus taxons.

Silkworm (Bombyx mori) hemocytes do not produce reactive oxygen metabolites as a part of defense mechanisms.

To investigate whether hemocytes of Bombyx mori (Lepidoptera) larvae produce reactive oxygen species (ROS) as part of the oxidative killing of invading pathogens, the production of ROS was measured as a luminol- and lucigenin-enhanced chemiluminescence of unstimulated or stimulated (zymosan particles, phorbol myristate acetate, calcium ionophore, rice starch or Xenorhabdus nematophila) hemolymph. No detectable ROS production was found. The spontaneous and activated ROS production measured with hemocytes, i.e. under the conditions when the antioxidative potential of hemolymph plasma was eliminated, was again undetectable. Likewise, ROS production by isolated hemocytes was observed by spectrophotometric (NBT test, cytochrome c assay) and fluorimetric (using dihydrorhodamine and hydroethidine probes) methods. Hence none of the experimental approaches used indicated the production of ROS by hemocytes of B. mori larvae as part of their immune response.