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PubMed1994

Crocidolite asbestos increased 8-hydroxydeoxyguanosine levels in cellular DNA of a human promyelocytic leukemia cell line, HL60.

Crocidolite, one of the most carcinogenic asbestos fibers, induces the release of reactive oxygen species (ROS) from neutrophils and macrophages. Using HPLC combined with electrochemical detection, we determined that 8-hydroxydeoxyguanosine (8OHdG), a molecule typical of mutagenic oxidative DNA damage, was induced in the cellular DNA of a human promyelocytic leukemia cell line, HL60, incubated with crocidolite. Crocidolite increased 8OHdG in the cellular DNA of phorbol myristate acetate (PMA)-differentiated HL60, which phagocytosed crocidolite. PMA-differentiated HL60 released ROS spontaneously, as determined by ESR with 5,5-dimethylpyrrolone-N-oxide as a spin trap. However, the release of ROS from the cell line did not increase after the addition of crocidolite. The addition of superoxide dismutase at a sufficient concentration to scavenge ROS released from the cell did not inhibit the 8OHdG increase induced by crocidolite. Cytochalasin B, which inhibited phagocytosis, did not inhibit the release of ROS. However, it inhibited the crocidolite-induced 8OHdG increase by 48.3%. Contrary to PMA-differentiated HL60, undifferentiated HL60 neither phagocytosed crocidolite nor showed a crocidolite-induced increase in 8OHdG formation. The 8OHdG increase induced by crocidolite was not correlated with ROS release, but with the internalization of crocidolite, suggesting that the increase was not due to an increase in ROS release from the cell but was due to the conversion of relatively inert ROS to highly reactive ROS, such as hydroxyl radicals, by crocidolite that was internalized and close to DNA.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2001

Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation.

BACKGROUND: Reactive oxygen species (ROS)-mediated damage to human spermatozoa has been implicated in the pathogenesis of male infertility. Although ROS production by human spermatozoa has been extensively studied, the cell-to-cell variation in ROS production by spermatozoa at different stages of maturation has never been investigated. METHODS: In this study, we determined ROS production by subsets of human spermatozoa at different stages of maturation isolated by density gradient centrifugation of ejaculated spermatozoa obtained from healthy donors and from patients attending a clinic for infertility screening. RESULTS: Four different fractions were obtained. ROS production was highest in immature spermatozoa with abnormal head morphology and cytoplasmic retention and lowest in mature spermatozoa and immature germ cells (P < 0.01). ROS production was highest in immature spermatozoa from males with abnormal semen parameters compared with donors (P < 0.0001) or patients with normal semen parameters (P = 0.015). ROS production by immature spermatozoa was inversely correlated with the recovery of motile, mature spermatozoa in the high density fraction 4 (P = 0.01). CONCLUSIONS: The results of this study indicate that there is significant cell-to-cell variation in ROS production in subsets of spermatozoa at different stages of maturation and that oxidative damage of mature spermatozoa by ROS-producing immature spermatozoa during sperm migration from the seminiferous tubules to the epididymis may be an important cause of male infertility.

Cellular Senescence↗

PubMedDate not supplied

Infections in the male genital tract and reactive oxygen species.

In the male genital tract, reactive oxygen species (ROS) are generated by spermatozoa and leukocytes including neutrophils and macrophages. ROS are involved in the regulation of sperm functions such as capacitation and the acrosome reaction. Infections lead to an excessive ROS production, resulting in an 'oxidative burst' from neutrophils/macrophages as a first-line defence mechanism. This is modulated by several cytokines and the pro-oxidant mechanisms of bacteria and viruses. At the site of an infection, the degree of activation of leukocytes, i.e. the amount of ROS produced, and the available antioxidative systems determine whether spermatozoa are damaged or not. During an infection, an imbalance of pro- and antioxidants favouring the former results in oxidative stress which impairs the sperm functions mentioned, as well as motility and fertilization. ROS produced during infections of the testis and epididymis are especially harmful to spermatozoa due to the longer contact time and the lack of antioxidant protection. In the final ejaculate, only very high numbers of ROS-producing leukocytes are detrimental to sperm functions. An infectious injury involving ROS in the prostate gland, seminal vesicles or epididymis could impair sperm functions indirectly. Pro- and antioxidative properties of therapeutics are currently receiving more attention as part of anti-infectious therapies. At present, there are many unresolved questions concerning the exact role of ROS during infections of the male genital tract because of the difficulty of specifically assessing the site of generation and the short-lived effects of ROS. New techniques may enable specific studies to fill this gap in the near future.

Antioxidants↗

PubMed2006

Antioxidants ameliorate the expression of vascular endothelial growth factor mediated by protein kinase C in diabetic podocytes.

BACKGROUND: The increased production of reactive oxygen species (ROS) may be involved in the onset or development of diabetic vascular complications. The release of ROS from podocytes plays a role in the pathogenesis of glomerular damage in various experimental glomerular diseases. Although it is assumed that the podocyte injury also plays an important role in diabetic nephropathy, the mechanism is still unknown. METHODS: Using a differentiated mouse podocyte cell line, we investigated: (1) whether a high level of ambient glucose increases the level of ROS, (2) whether the protein kinase C (PKC) pathway is involved in a high-glucose-induced generation of ROS and vascular endothelial growth factor (VEGF) and (3) whether antioxidants ameliorate PKC-mediated VEGF expression in diabetic milieu. RESULTS: Intracellular ROS generation was significantly higher in high glucose than in control conditions in cultured podocytes. High ambient glucose also increased VEGF mRNA and protein expression. The high-glucose-induced increases in ROS and VEGF mRNA and protein by podocytes were effectively inhibited by pretreatment with various antioxidants and were completely restored by PKC inhibition. The results show that cultured mouse podocytes produce ROS in response to high glucose, and that PKC is involved in high-glucose-induced ROS and VEGF production by podocyte. CONCLUSION: Increased ROS in podocytes may play a role in the pathogenesis of podocyte injury in diabetic nephropathy.

Animals↗

PubMed2003

Similar stress responses are elicited by copper and ultraviolet radiation in the aquatic plant Lemna gibba: implication of reactive oxygen species as common signals.

Metals and ultraviolet (UV) radiation are two environmental stressors that can cause damage to plants. These two types of stressors often impact simultaneously on plants and both are known to promote reactive oxygen species (ROS) production. However, little information is available on the potential parallel stress responses elicited by metals and UV radiation. Using the aquatic plant Lemna gibba, we found that copper and simulated solar radiation (SSR, a light source containing photosynthetically active radiation (PAR) and UV radiation) induced similar responses in the plants. Both copper and SSR caused ROS formation. The ROS levels were higher when copper was combined with SSR than when applied with PAR. Higher concentrations of copper plus PAR caused toxicity as monitored by diminished growth and chlorophyll content. This toxicity was more pronounced when copper was combined with SSR. Because the generation of ROS was also higher when copper was combined with SSR, we attributed this enhanced toxicity to elevated levels of ROS. In comparison to PAR-grown plants, SSR treated plants exhibited elevated levels of superoxide dismutase (SOD) and glutathione reductase (GR). These enzyme levels were further elevated under both PAR and SSR when copper was added at concentrations that generated ROS. Interestingly, copper treatment in the absence of SSR (i.e. copper plus PAR) induced synthesis of the same flavonoids as those observed in SSR without copper. Finally, addition of either dimethyl thiourea or GSH (two common ROS scavengers) lowered in vivo ROS production, alleviated toxicity and diminished induction of GR as well as accumulation of UV absorbing compounds. Thus, the potential of ROS being a common signal for acclimation to stress by both copper and UV can be considered.

Acyltransferases↗

PubMed2002

Silica-induced generation of extracellular factor(s) increases reactive oxygen species in human bronchial epithelial cells.

Chronic inflammation and production of DNA-damaging reactive oxygen species (ROS) may be involved in silica-induced lung cancer. Studies to date have largely focused on silica-induced production of ROS by lung phagocytes. In this study, we investigated the hypothesis that particulate silica (DQ12) can also induce elevations in intracellular ROS in a cancer-target cell type, i.e., human bronchial epithelial cells (BECs), via an indirect mechanism that involves ROS-inducing extracellular factor(s) that occur upon the interaction of silica with culture medium. The intracellular production of hydrogen peroxide (H(2)O(2)) in BECs was assessed by flow cytometry via monitoring dichlorofluorescein (DCF) fluorescence. Culture medium containing 10% human serum was incubated with silica particles in concentrations ranging from 10 to 50 microg/ml, and following incubation for 1 h and removal of the particles, the resulting supernatants were added to BECs. Silica-treated medium induced significant increases in intracellular H(2)O(2) after the medium had been treated with as little as 10 microg/ml of the particles. Further, the level of ROS increases in BECs in response to silica-treated medium was found to be virtually identical to that induced in cells that were directly treated with silica in suspension. Based on enzyme inhibitory studies, the mechanism for this increased generation of intracellular ROS appears to involve both mitochondrial respiration and a NAD(P)H oxidase-like system. Spectrofluorimetric experiments with the antioxidant enzymes superoxide dismutase and catalase showed that superoxide anions (O2*-) and H(2)O(2) are generated in silica-treated medium, but these ROS do not fully account for the induction of the intracellular ROS response. Iron, on the other hand, was found to be crucial to the process. Our collective results suggest silica-aqueous medium interactions can lead to the generation of factor(s) that induce the intracellular production of potentially DNA-damaging ROS in BECs in a manner that does not require direct particle-cell interactions.

Bronchi↗

PubMed1997

Protein kinase C- and Ca2+ ionophore-stimulated production of reactive oxygen species in mechanically dispersed isolated bovine luteal cells.

We measured the production of reactive oxygen species (ROS) using luminol-horseradish peroxidase-induced chemiluminescence in mechanically dispersed cell suspensions from bovine corpus luteum (CL). Since other cell types besides luteal cells were present in crude cell suspensions from CL, cell preparations were purified by centrifugation on Percoll. Only cell suspensions that gave no significant response when stimulated with formyl-methionyl-leucyl-phenylalanine, a potent stimulator of ROS production by phagocytes, were used routinely. Basal ROS production by purified bovine luteal cell preparations was low but could be stimulated rapidly and in a dose-dependent manner by nanomolar concentrations of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), though only in cells purified by fractionation on Percoll. Luteal ROS responses to PMA were quenched by returning bovine erythrocytes to purified luteal cells, or by exogenous catalase or superoxide dismutase. The magnitude of the response to PMA varied markedly from one luteal cell preparation to another but appeared to be unrelated to the stage of the luteal phase of the CL from which the cells were prepared. The luteal ROS response to PMA was blocked by staurosporine, an inhibitor of PKC. Although the inactive phorbol ester (4alpha-phorbol didecanoate; 4alphaPDD) alone had little or no effect on luteal ROS production, 4alphaPDD significantly potentiated the effects of submaximal concentrations of PMA in a dose-dependent manner. ROS production could also be stimulated by the Ca2+ ionophore A23187. This response was rapidly abolished by treatment with EDTA or EGTA. A23187 also augmented the response to submaximal PMA levels: however, pretreatment with 4alphaPDD did not significantly enhance the ROS response to A23187. In conclusion, we have shown that isolated bovine luteal cell suspensions are capable of generating a marked acute ROS response triggered by activation of PKC and/or elevation of cytosolic calcium.

Animals↗

PubMed2003

Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria.

Several lines of evidence indicate that mitochondria-mediated reactive oxygen species (ROS) generation is a major source of oxidative stress in the cell. Release of cytochrome c from mitochondria is a central event in apoptosis induction and appears to be mediated by ROS. Dissociation of cytochrome c from the IMM, where it is bound to cardiolipin, represents a necessary first step for cytochrome c release. In the present study, the role of ROS and cardiolipin in the release of cytochrome c from rat liver mitochondria was investigated. ROS were produced by mitochondria oxidizing succinate in the nonphosphorylating state. Cytochrome c was quantitated by a new, very sensitive and rapid reverse-phase HPLC method. We found that succinate-supported ROS production resulted in a release of cytochrome c from mitochondria and a parallel loss of cardiolipin content. These effects were directly and significantly correlated and also abolished by ADP, which prevents succinate-mediated ROS production. The ROS-induced cytochrome c release was independent from MPT and appears to involve VDAC. It is suggested that mitochondrial-induced ROS production promotes cytochrome c release from mitochondria by a two-steps process, consisting of the dissociation of this protein from cardiolipin, followed by permeabilization of the outer membrane, probably by interaction with VDAC. The data may help clarify the molecular mechanism underlying the release of cytochrome c from the mitochondria to the cytosol and the role of ROS and cardiolipin in this release.

Animals↗

PubMed2000

Rac1 inhibits TNF-alpha-induced endothelial cell apoptosis: dual regulation by reactive oxygen species.

Reactive oxygen species (ROS) have been implicated as mediators of tumor necrosis factor-alpha (TNF) -induced apoptosis. In addition to leading to cell death, ROS can also promote cell growth and/or survival. We investigated these two roles of ROS in TNF-induced endothelial cell apoptosis. Human umbilical vein endothelial cells (HUVECs) stimulated with TNF produced an intracellular burst of ROS. Adenoviral-mediated gene transfer of a dominant negative form of the small GTPase Rac1 (Rac1N17) partially suppressed the TNF-induced oxidative burst without affecting TNF-induced mitochondrial ROS production. HUVECs were protected from TNF-induced apoptosis. Expression of Rac1N17 blocked TNF-induced activation of nuclear factor-kappa B (NF-kappaB), increased activity of caspase-3, and markedly augmented endothelial cell susceptibility to TNF-induced apoptosis. Direct inhibition of NF-kappaB through adenoviral expression of the super repressor form of inhibitor of kappaBalpha (I-kappaB S32/36A) also increased susceptibility of HUVECs to TNF-induced apoptosis. Rotenone, a mitochondrial electron transport chain inhibitor, suppressed TNF-induced mitochondrial ROS production, proteolytic cleavage of procaspase-3, and apoptosis. These findings show that Rac1 is an important regulator of TNF-induced ROS production in endothelial cells. Moreover, they suggest that Rac1-dependent ROS, directly or indirectly, lead to protection against TNF-induced death, whereas mitochondrial-derived ROS promote TNF-induced apoptosis.

Adenoviridae↗

PubMed1998

Increased formation of reactive oxygen species after permanent and reversible middle cerebral artery occlusion in the rat.

In barbiturate-anesthetized rats, we induced 3 hours of permanent middle cerebral artery occlusion (MCAO) by an intraluminal thread (n = 6), or 1 hour MCAO followed by 2 hours of reperfusion (n = 6). Through a closed cranial window over the parietal cortex, the production of reactive oxygen species (ROS) was measured in the infarct border using online in vivo chemiluminescence (CL) while monitoring the appearance of peri-infarct depolarizations (PID). The borderzone localization of the ROS and direct current (DC) potential measurements was confirmed in additional experiments using laser-Doppler scanning, mapping regional CBF changes through the cranial window after permanent (n = 5) or reversible (n = 5) MCAO. CL measurements revealed a short period (10 to 30 minutes) of reduced ROS formation after vessel occlusion, followed by a significant increase (to 162 +/- 51%; baseline = 100%; P < .05) from 100 minutes of permanent MCAO onward. Reperfusion after a 1-hour period of MCAO led to a burst-like pattern of ROS production (peak: 489 +/- 330%; P < .05). When the experiments were terminated 3 hours after induction of MCAO, CL was still significantly increased above baseline after permanent and reversible MCAO (to 190 +/- 67% and 211 +/- 64%, respectively; P < .05). Simultaneous DC potential recordings detected 6.4 +/- 2.7 PID in the first, 4.7 +/- 2.3 in the second, and 2.8 +/- 2.0 in the third hour after permanent MCAO. In animals with reversible MCAO, PID were abolished from 15-minutes recirculation onward. There was no temporal relationship between ROS production and peri-infarct DC potential shifts. In conclusion, using a high temporal resolution ROS detection technique (CL), we found that permanent MCAO (after an initial decrease) was accompanied by a steady increase of ROS production during the 3-hour observation period, while reperfusion after 1 hour of MCAO produced a burst in ROS formation. Both patterns of ROS production were not related to the occurrence of PID.

Acridines↗

PubMed2004

Time-dependent transition of tempol-sensitive reduction of blood pressure in angiotensin II-induced hypertension.

OBJECTIVE: Reactive oxygen species (ROS) participate in the intracellular signalling of angiotensin II. However, the mechanisms of the interaction of ROS with hypertension and mitogen-activated protein kinase (MAPK) in vivo have remained unclear. Angiotensin II infusion provokes sustained hypertension accompanied with enhancement of ROS production; initially hypertension is non-sensitive to ROS, but thereafter becomes sensitive. We examined the time-dependent transition of ROS-sensitive vasoconstriction during angiotensin II infusion and also ROS sensitivity to cardiovascular MAPK activation in acutely and chronically angiotensin II-infused rats. METHODS AND RESULTS: During infusion of a pressor dose of angiotensin II to conscious Sprague-Dawley rats, tempol, a superoxide dismutase mimetic, was administered at 10 min, some 1, 3, 6, 12 and 24 h after the start of infusion. The magnitude of the reduction in blood pressure by tempol was initially negligible, but gradually enlarged, and reached a maximum of 96% of delta increase by angiotensin II at 12 h. However, even after sensitization to tempol, superimposed angiotensin II enabled an increase of blood pressure under tempol treatment. In chronically angiotensin II-infused rats, superimposed angiotensin II exhibited tempol quenchable MAPK activation. CONCLUSIONS: These results indicate that the mechanisms of angiotensin II-induced vasoconstriction may shift from being non-sensitive to ROS to sensitive within 12 h; nevertheless, both ROS non-sensitive vasoconstriction and ROS-sensitive MAPK activation by angiotensin II, which are both seen in the acute phase of infusion, are restored in the late maintaining phase of prolonged angiotensin II infusion.

Angiotensin II↗

PubMed2004

Activation of the signal transducer and activator of transcription signaling pathway in renal proximal tubular cells by albumin.

Renal proximal tubular cells activated by reabsorption of protein are thought to play significant roles in the progression of kidney diseases. It was hypothesized that the signal transducer and activator of transcription (STAT) proteins may be activated by proteinuria in proximal tubular cells. To test this hypothesis, murine proximal tubular cells were treated with albumin (30 mg/ml medium) for various lengths of time. The results showed that albumin could activate Stat1 and Stat5 within 15 min in proximal tubular cells. The activation of STATs was mediated mostly by Jak2 and required no protein synthesis. In addition, activation of Stat1 occurred even after neutralization of IFN-gamma. The activation of STATs was inhibited by N-acetyl-L-cysteine, a precursor of glutathione and a reactive oxygen species (ROS) scavenger, and fluorescence-activated cell sorter analysis showed upregulation of intracellular ROS after albumin overloading, suggesting that albumin per se could generate ROS in proximal tubular cells. The activation of STATs occurred by way of the ROS generating system, and especially through the membrane-bound NADPH oxidase system. Reduced activities of glutathione peroxidase and catalase could also be responsible for the accumulation of intracellular ROS. Hence, not only the ROS generating system, but also the ROS scavenging system may contribute to the induction of ROS by albumin. These findings support the hypothesis that proximal tubular cells are activated and generate ROS by reabsorption of abundant urinary proteins filtered through the glomerular capillaries, and as a consequence, various IFN-gamma-inducible proteins are synthesized through IFN-gamma-independent activation of STAT signaling.

Albumins↗

PubMed2004

Phosphatidic acid induces leaf cell death in Arabidopsis by activating the Rho-related small G protein GTPase-mediated pathway of reactive oxygen species generation.

Phosphatidic acid (PA) level increases during various stress conditions. However, the physiological roles of this lipid in stress response remain largely unknown. In this study, we report that PA induced leaf cell death and elevated the levels of reactive oxygen species (ROS) in the whole leaf and single cells. To further elucidate the mechanism of PA-induced cell death, we then examined whether Rho-related small G protein (ROP) 2, which enhanced ROS production in an in vitro assay, is involved in PA-induced ROS production and cell death. In response to PA, transgenic leaves of Arabidopsis expressing a constitutively active rop2 mutant exhibited earlier cell death and higher levels of ROS than wild type (WT), whereas those expressing a dominant-negative rop2 mutant exhibited later cell death and lower ROS. However, in the absence of exogenous PA, no spontaneous cell death or elevated ROS was observed in constitutively active rop2 plants, suggesting that the activation of ROP GTPase alone is insufficient to activate the ROP-mediated ROS generation pathway. These results suggest that PA modulates an additional factor required for the active ROP-mediated ROS generation pathway. Therefore, PA may be an important regulator of ROP-regulated ROS generation and the cell death process during various stress and defense responses of plants.

Apoptosis↗

PubMed2007

Hyperactivity and reactivity of peripheral blood neutrophils in chronic periodontitis.

Some evidence exists that peripheral neutrophils from patients with chronic periodontitis generate higher levels of reactive oxygen species (ROS) after Fcgamma-receptor stimulation than those from healthy controls. We hypothesized that peripheral neutrophils in periodontitis also show both hyper-reactivity to plaque organisms and hyperactivity in terms of baseline, unstimulated generation and release of ROS. Peripheral neutrophils from chronic periodontitis patients and age/sex/smoking-matched healthy controls (18 pairs) were assayed for total ROS generation and extracellular ROS release, with and without stimulation (Fcgamma-receptor and Fusobacterium nucleatum), using luminol and isoluminol chemiluminescence. Assays were performed with and without priming with Escherichia coli lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Phox gene expression (p22, p47, p67, gp91) was investigated using reverse transcription-polymerase chain reaction (RT-PCR). Neutrophils from patients produced higher mean levels of ROS in all assays. Total generation and extracellular release of ROS by patients' cells were significantly greater than those from controls after FcgammaR-stimulation, with (P = 0.023) and without (P < or = 0.023) priming with GM-CSF. Differences in unstimulated total ROS generation were not significant. By contrast, patients' cells demonstrated greater baseline, extracellular ROS release than those from controls (P = 0.004). This difference was maintained after priming with LPS (P = 0.028) but not GM-CSF (P = 0.217). Phox gene expression was similar in patient and control cells at baseline and stimulation with F. nucleatum (3 h) consistently reduced gp91(PHOX) transcripts. Our data demonstrate that peripheral neutrophils from periodontitis patients exhibit hyper-reactivity following stimulation (Fcgamma-receptor and F. nucleatum) and hyperactivity in terms of excess ROS release in the absence of exogenous stimulation. This hyperactive/-reactive neutrophil phenotype is not associated with elevated phox gene expression.

Adult↗

PubMed1997

Inhibitory effect of local anaesthetics on reactive oxygen species production by human neutrophils.

BACKGROUND: Reactive oxygen species (ROS) generated from neutrophils accumulated in various major organs are thought to play a pivotal role in the pathogenesis of host auto-injury. Lidocaine has been shown to reduce the injury. We investigated the effect of local anaesthetics (lidocaine, mepivacaine and bupivacaine) on ROS production by neutrophils using an in vitro system. METHODS: We measured the production of superoxide (ferricytochrome c method), hydrogen peroxide (H2O2: scopoletin fluorescence technique), and hydroxyl radical (OH.: ethylene gas method) by neutrophils isolated from human adult volunteers in the absence and presence of lidocaine (2-200 micrograms/mL), mepivacaine (3-300 micrograms/mL), and bupivacaine (3-300 micrograms/mL). We also measured the ROS generation in a cell-free (xanthine-xanthine oxidase) system. RESULTS: Lidocaine and mepivacaine at higher levels significantly decreased the production of ROS by neutrophils. However, these local anaesthetics at clinically relevant blood concentrations had no effect on the levels of ROS. Furthermore, lidocaine and mepivacaine failed to reduce ROS generated by the cell-free system. Bupivacaine did not decrease ROS generation by either generating system. CONCLUSION: In conclusion, in the present in vitro system, only concentrations of lidocaine and mepivacaine 100-fold higher than clinically feasible ones reduced ROS production by human neutrophils. However, the local anaesthetics at clinically relevant blood concentrations had no suppressive effect. Further studies using in vivo systems are required to elucidate the inhibitory effects of local anaesthetics on ROS generation in clinical settings.

Adult↗

PubMed2005

Alpha-1-acid glycoprotein inhibits phorbol ester-induced but not Fc-receptor-induced generation of reactive oxygen species in bovine peripheral blood neutrophils.

Alpha-1-acid glycoprotein (AGP) is an acute-phase protein with anti-inflammatory and immunomodulating properties. AGP is described as a potent inhibitor of the production of reactive oxygen species (ROS) in human neutrophils. However, published reports about the mechanism of inhibition are conflicting. The influence of bovine AGP on the production of ROS by bovine peripheral blood polymorphonuclear leucocytes (PMN) was studied using a highly sensitive method approaching its inhibitory mechanism. ROS production in PMN was induced with phorbol 12-myristate 13-acetate (PMA) or opsonized Staphylococcus aureus bacteria. ROS generation was quantified and evaluated by flow cytometry. AGP efficiently suppressed PMA, but did not opsonize bacteria-induced ROS generation in vitro. The suppressive effect was concentration-dependent and adversely proportional to PMA concentration. The selective inhibitory potential of AGP in comparison with ovalbumin (OVA) and bovine serum albumin (BSA) showed that ROS inhibition was not a mere protein effect. ROS production was suppressed only if AGP and PMA were simultaneously present with PMN. Pre-incubation of PMN with AGP did not alter the PMN response to PMA. Moreover, AGP could not suppress ROS production after pre-stimulation of PMN with PMA. Human and bovine AGP did not differ in their inhibitory potential to the PMA-induced ROS production in bovine, human and equine PMN. The results show that AGP does not modulate bovine neutrophil functions directly, but acts as a scavenger of PMA.

Animals↗

PubMed2000

Mitochondrial function and antioxidative defence in human muscle: effects of endurance training and oxidative stress.

The influence of endurance training on oxidative phosphorylation and the susceptibility of mitochondrial oxidative function to reactive oxygen species (ROS) was investigated in skeletal muscle of four men and four women. Mitochondria were isolated from muscle biopsies taken before and after 6 weeks of endurance training. Mitochondrial respiration was measured before and after exposure of mitochondria to exogenous ROS (H2O2 + FeCl2). Endurance training increased peak pulmonary O2 uptake (VO2,peak) by 24 % and maximal ADP-stimulated mitochondrial oxygen consumption (state 3) by 40% (P<0.05). Respiration in the absence of ADP (state 4), the respiratory control ratio (RCR = state 3/state 4) and the ratio between added ADP and consumed oxygen (P/O) remained unchanged by the training programme. Exposure to ROS reduced state 3 respiration but the effect was not significantly different between pre- and post-training samples. State 4 oxygen consumption increased after exposure to ROS both before (+189 %, P< 0.05) and after training (+243 %, P<0.05) and the effect was significantly higher after training (P<0.05, pre- vs. post-training). The augmented state 4 respiration could in part be attenuated by atractyloside, which indicates that ADP/ATP translocase was affected by ROS. The P/O ratio in ROS-treated mitochondria was significantly lower (P<0.05) compared to control conditions, both before (-18.6+/-2.2 %) and after training (-18.5+/-1.1%). Muscle activities of superoxide dismutase (mitochondrial and cytosolic), glutathione peroxidase and muscle glutathione status were unaffected by training. There was a positive correlation between muscle superoxide dismutase activity and age (r = 0.75; P<0.05; range of age 20-37 years), which may reflect an adaptation to increased generation of ROS in senescent muscle. The muscle glutathione pool was more reduced in subjects with high activity of glutathione peroxidase (r = 0.81; P<0.05). The influence of short-term training on mitochondrial oxygen consumption has for the first time been investigated in human skeletal muscle. The results showed that maximal mitochondrial oxidative power is increased after endurance training but that the efficiency of energy transfer (P/O ratio) remained unchanged. Antioxidative defence was unchanged after training when expressed relative to muscle weight. Although this corresponds to a reduced antioxidant protection per individual mitochondrion, the sensitivity of aerobic energy transfer to ROS was unchanged. However, the augmented ROS-induced non-coupled respiration after training indicates an increased susceptibility of mitochondrial membrane proton conductance to oxidative stress.

Adult↗

PubMed2007

The enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis is enhanced under conditions where reactive oxygen species are neutralized.

AIM: To investigate the effect of neutralization of reactive oxygen species (ROS-neutralized conditions) on the enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis using selective and nonselective media. METHODS: Pure cultures of E. coli NCTC8912 and Ent. faecalis NCTC775 were injured using dilute sodium hypochlorite, at free chlorine levels of 0.6 and 0.9 microg ml(-1), respectively, and then enumerated at 37 degrees C by surface plate counts on nonselective nutrient (N) agar and on several selective media, either under (i) standard aerobic conditions; (ii) aerobic conditions using growth medium, supplemented with 0.05%-w/v sodium pyruvate, to neutralize peroxides; or (iii) conditions designed to neutralize ROS, using a combination of 0.05%-w/v sodium pyruvate in the growth medium, together with incubation in an anaerobic jar. RESULTS: The counts obtained on the nonselective medium were lowest under aerobic conditions in unsupplemented medium, higher in pyruvate-supplemented (peroxide-neutralized) medium and highest for ROS-neutralized conditions. Counts for the selective media were often lower than those for nonselective N (nutrient) agar, with enhancement under peroxide-neutralized conditions and a further increase in counts under ROS-neutralized conditions. Broadly similar observations were made for three other strains of each organism. CONCLUSIONS: Chlorine-injured E. coli and Ent. faecalis become sensitive to ROS, giving higher counts under ROS-neutralized enumeration conditions than under conventional aerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The enhancement in counts observed under ROS-neutralized conditions indicate that the addition of pyruvate to the growth medium may not fully counteract the effects of sublethal injury under aerobic conditions, which is a novel observation. Thus, ROS-neutralized conditions may be required for optimal enumeration of faecal indicator bacteria. Furthermore, the lower counts, obtained using selective media indicate that the sensitivity of chlorine-injured bacteria to selective agents is not necessarily reversed under ROS-neutralized conditions.

Bacteriological Techniques↗