Cholinergic-induced production of reactive oxygen species in human neuroblastoma cells

Stimulation of human SH-SY5Y neuroblastoma cells by a muscarinic receptor agonist, carbachol (CCh; 1 mM), elevated levels of free intracellular calcium and subsequently increased the production of reactive oxygen species (ROS). Quinuclidinylbenzilate (QNB) binding increased at 1 h after CCh, but returned back to the control level at 3 h. Production of ROS increased, however, during the 3 h time period. CCh also increased the translocation of protein kinase C (PKC) to the membrane. ROS production was completely blocked by atropine and a PKC inhibitor, Ro 31-8220. These results show that increased ROS production was a result of muscarinic receptor stimulation, and that PKC had an active role in this cellular stimulation. ROS production upon cellular stimulation by CCh was completely inhibited also by superoxide dismutase, and partially by catalase, indicating that the formation of superoxide anion dominated in cholinergic-induced generation of ROS in human neuroblastoma cells. These results also show that muscarinic stimulation causes sustained ROS production in human neuroblastoma cells. The slow increase in ROS production by CCh suggest a stepwise cascade of events leading to oxidative stress with a triggering role of cholinergic muscarinic receptors in this process.     

Zinc aspartate in vivo and in vitro modulation of reactive oxygen species production by human neutrophils and monocytes

In April 1991, an oral health situation analysis team comprising oral health professionals from the Alaska Area Native Health Service, the World Health Organization, and the Moscow Medical Stomatological Institute visited the Magadan region of the Soviet Far East at the request of the Ministries of Health of both the Russian Republic and the then-Soviet Union. As few oral health data had been available specific to the Magadan region, the purpose of the trip was to gather data concerning oral diseases epidemiology, demography, and health systems of the communities of Magadan, Seymchan, and Provideniya as the basis for recommendations and plan development. The data were collected using standard WHO methodology. The analysis team examined children in the age cohorts of 3-5, 6-7, 12, and 15 years. In addition, adults aged 35-44 and 65-74 years were examined in Magadan and Seymchan. Both caries and periodontal rates are high, with a rate of 4.3 DMFT among 12-year-olds in the total region, but up to 5.4 DMFT in one location. Specific findings and recommendations have been forwarded to local community and health officials. The project is ongoing, and plans are underway to provide continuing assistance in designing and implementing an effective program for the prevention and treatment of oral diseases.     

Effect of culture, incubation and acrosome reaction of fresh and frozen-thawed ram spermatozoa for in vitro fertilization and intracytoplasmic sperm injection

This study evaluated different sperm treatments for fertilization of sheep oocytes by intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF). In Experiment 1, fresh and frozen semen was separated by Percoll centrifugation and incubated at 30 degrees C or 39 degrees C in HSOF or BSOF medium for 1 h before use for IVF or ICSI. For IVF, oocytes were inseminated and incubated with sperm for 30 min, 4 h and 19 h. Sperm were assessed for acrosome integrity after Percoll centrifugation and 1 h incubation, and those used for IVF were assessed after each period of exposure to the oocytes. Fertilization rates after ICSI were higher for fresh than for frozen-thawed sperm and were highest 19 h after IVF with fresh or frozen-thawed sperm in the presence of HSOF at 30 degrees C. In Experiment 2, fresh semen was separated by Percoll centrifugation and incubated for 5 h in HSOF, and the acrosome reaction was induced with lysophosphatidylcholine. Acrosome integrity was then assessed. Fertilization rates after ICSI were similar for acrosome-reacted and control spermatozoa. These results suggest that induction of the acrosome reaction in spermatozoa before ICSI is unnecessary, whereas a capacitating treatment of spermatozoa is required before IVF.     

Direct effects of reactive oxygen species on cochlear outer hair cell shape in vitro

Reactive oxygen species (ROS) have been implicated in the ototoxicity of various agents. This study examines the effects of superoxide anion (O2), hydroxyl radical (OH.) and hydrogen peroxide (H2O2), on isolated cochlear outer hair cell (OHC) morphology. OHCs were superfused with artificial perilymph (AP) or AP containing a specific ROS scavenger, and then with AP, ROS system or scavenger plus ROS system for 90 min. The generation of ROS as well as the scavenging properties of other agents were confirmed by specific biochemical assays. Control cells decreased 4.8% in mean length, and showed no obvious membrane damage. Generation of O2. or OH. resulted in high rates (85.7 and 42.9%, respectively) of bleb formation at the synaptic pole, and decreased (O2., 15.2%; OH., 17.3%) mean cell length. Length change and bleb formation rate were H2O2 concentration-dependent. 20 mM H2O2 led to 33.3% decreased mean cell length, and only 20% bleb formation; 0.1 mM H2O2 led to 83.3% bleb formation, with no length decrease. Superoxide dismutase, deferoxamine and catalase protected against O2., OH. and H2O2 effects, respectively. Bleb formation and diminished cell length likely represent differential lipid peroxidative outcomes at supra- and infranuclear membranes, and are consistent with effects of certain ototoxicants.     

Formation of reactive oxygen species by pentaerithrityltetranitrate and glyceryl trinitrate in vitro and development of nitrate tolerance

Anti-ischemic therapy with organic nitrates is complicated by tolerance. Induction of tolerance is incompletely understood and likely multifactorial. Recently, increased production of reactive oxygen species (ROS) has been investigated, but it has not been clear if this is a direct consequence of the organic nitrate on the vessel or an in vivo adaptation to the drugs. To examine the possibility that nitrates could directly stimulate vascular ROS production, we compared the development of nitrate tolerance with the formation of ROS induced by pentaerithrityltetranitrate (PETN) or nitroglycerin (GTN) in vitro in porcine smooth muscle cells, endothelial cells, washed ex vivo platelets and whole blood. By examining cGMP formation, it was found that 24-hr treatment with GTN but not PETN induced significant nitrate tolerance, which was prevented by parallel treatment with Vit C. Incubation of vascular cells acutely with 0.5 mM GTN doubled the rate of ROS generation, whereas PETN had no such effect. The rate of ROS (peroxynitrite and O2) formation detected by specific spin traps in tolerant smooth muscle cells, treated for 24 hr with 0.01 mM GTN, was substantially higher (30.5 nM/min) than in control cells acutely treated with 0.5 mM GTN (25 nM/min). In contrast to PETN, GTN induces nitrate tolerance and also increases the formation of ROS both in vascular cells and in whole blood. ROS formation is minimally stimulated by PETN comparable to data obtained in Vit C-suppressed GTN tolerance. ROS formation induced by organic nitrates seems to be a key factor in the development of nitrate tolerance.     

Pramipexole reduces reactive oxygen species production in vivo and in vitro and inhibits the mitochondrial permeability transition produced by the parkinsonian neurotoxin methylpyridinium ion

CeReS-18 is a unique negative regulator of cell proliferation with a wide array of target cells. To elucidate the mechanism by which CeReS-18 mediates cell growth inhibition, the possibility that CeReS-18 alters the function of G1 cyclins and their respective cyclin-dependent kinases (cdks) has been examined in mouse fibroblasts (Swiss 3T3) synchronized by CeReS-18. We show here that cyclin D-associated cdk activity is significantly inhibited in the CeReS-18-treated cells. Corresponding to the inhibited cdk function, we demonstrate a low expression of cyclin D in mid G1 determined by Western blot analysis, and cyclin D was greatly reduced in the immunocomplex recovered with antibody to cdk4 and cdk6. Previously, we have shown that the retinoblastoma susceptibility gene product (pRb), a key substrate of cyclin D-cdk complex, was maintained in the hypophosphorylated state in the CeReS-18-inhibited cells. We conclude here that cyclin D/cdk4,6/pRb is the major pathway by which CeReS-18 mediates cell cycle arrest.     

Effects of gamma-aminobutyric acid, progesterone and ionophore A23187 on acrosome reaction of tree shrew sperm in vitro: examination of acrosome reaction with an improved fluorescence microscopy

A number of acrosome reaction (AR) initiators have been found to be effective in inducing AR of human, laboratory and domestic animal sperm. Using an improved simple fluorescence microscopy, effects of gamma-aminobutyric acid (GABA), progesterone and ionophore A23187 on sperm AR of tree shrew, a useful animal model in biomedical research, have been investigated. Spontaneous AR in 4.92-7.53% of viable sperm was observed. Complete AR in 10.31-18.25% of viable tree shrew sperm was obviously induced by 5 microM and 10 microM calcium ionophore A23187, 1 mM GABA, and 5 microM progesterone, and there were no significant differences between their abilities to initiate complete AR. No significant differences of AR percentages between 1- and 2-h treatments with A23187, progesterone and/or GABA were observed. These results suggested that the responses of tree shrew sperm to these AR initiators are similar to that of human and other mammalian sperm.     

Effect of in vitro incubation on spontaneous acrosome reaction in fresh and cryopreserved human spermatozoa

OBJECTIVES: Capacitation and acrosome reaction are prerequisites for fertilization. However, in vitro capacitation is not necessary for an agonist-induced acrosome reaction. We studied whether in vitro capacitation is important in spontaneous acrosome reaction and analyzed how capacitation before cryopreservation influences the acrosomal status of thawed spermatozoa. METHODS: Semen specimens from normal donors (n = 15) were processed by the swim-up technique and divided into two aliquots. One aliquot was capacitated (capacitation induced) for three hours by incubation in a modified-BWW medium with 3% HSA at 37 degrees C under 5% carbon dioxide. The other aliquot did not receive any treatment. Both aliquots were analyzed by CASA to assess the capacitation status of the spermatozoa and then cryopreserved. Spontaneous acrosome reaction was assessed by FITC-PNA lectin before and after cryopreservation. Sperm viability was measured using Hoechst-33258 stain. RESULTS: Before freezing, the frequency of spontaneous acrosome reaction was higher in the capacitation-induced sperm preparation (median, 20.5% [interquartile range, 17.2-37.8]) than in swim-up-induced specimens (median, 10.6% [range, 4.8-23.2]; P <.001). The percentage of viable cells showing acrosome reaction increased after cryopreservation in both swim-up-induced specimens (median, 241.4% [interquartile range, 37.1-678.6]; P <.001) and capacitation-induced specimens (median, 48.2% [range, 6.1-63.3]; P = 0.002). Although this increase was higher in the swim-up-induced specimens (P = 0.002), frequency of postthaw spontaneous acrosome reaction was similar in both groups (P = 0.18). CONCLUSIONS: We conclude that sperm capacitation significantly optimizes the acrosome reaction. However, a small proportion of normal spermatozoa do not require capacitation to undergo spontaneous acrosome reaction in vitro. After cryopreservation, the percentage of spermatozoa that had intact acrosomes was similar in both groups, despite the fact that one aliquot underwent prefreeze capacitation. These findings suggest that the acrosome reaction may involve complex mechanism(s) rather than a physiological change induced by capacitation.     

Flow cytometric analysis of DNA and nuclear protein in paraffin-embedded tissue

Human insulin-like growth factor I (IGF1) was labeled with 125I and the resulting mixture of iodination isomers was separated by reverse-phase HPLC. Three major radioactive peaks were isolated and identified by sequencing as the expected three monoiodinated species. The ranking of the affinities of the three isomers for the human IGF1 receptor was found to be Tyr24(125I) > Tyr31(125I) > Tyr60(125I). The Tyr31(125I) isomer was shown to have an affinity similar to that of unlabeled IGF1 and is thus the tracer of choice for IGF1. The tracers were stable upon storage at -20 degrees C for at least 3 months.     

Interferon beta-1b inhibits reactive oxygen species production in peripheral blood monocytes of patients with relapsing-remitting multiple sclerosis

To determine if perforation rate is a function of delayed diagnosis or delayed presentation in childhood acute appendicitis we performed a retrospective casenote review of 101 consecutive children undergoing emergency appendicectomy over a 12-month period. The perforation rate was 7% in those children presenting with symptoms of 1 day or less and was significantly greater (33%, Chi 2 = 9.45, P < 0.01) in those who had had symptoms for more than 1 day at presentation. There was no difference in in-hospital delay between the groups. A high perforation rate was found to be a feature of delayed presentation.     

Effects of acute endurance exercise and 8 week training on the production of reactive oxygen species from neutrophils in untrained men

The suboptimal efficacy of the currently available 23-valent pneumococcal vaccine in the growing population of adults >65 years old may be related to the limited immunogenicity of the vaccine polysaccharides in this group. In this study, the majority of elderly outpatients with stable chronic illnesses generated a vigorous IgG response to seven vaccine serotypes comparable to that of healthy young adults at 1, 3, and 16 months after immunization. Moreover, the quality and function of anticapsular antibodies, measured as avidity and in vitro opsonization, were comparable between elderly and young subjects over time. However, a subset (approximately 20%) of elderly outpatients responded to fewer than two of seven serotypes tested 1 and 3 months after immunization, whereas none of the healthy young adults were such poor responders. Thus, despite the adequate mean immune responses of the elderly as a group, a substantial proportion of elderly persons may have poor responses to the currently available pneumococcal vaccine.     

Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species

This article describes the use and prescribing of menopausal and postmenopausal hormone therapy (HT) in one example country, Finland, and the trends and levels of HT use in other western countries for comparison. Previously published studies were reviewed and reanalyzed, and some additional unpublished data from Finnish surveys were compiled. The use of HT increased in Finland up to 1994. In Finland the initiative for HT use came more often from physicians than women themselves, physicians valued HT more than women, women's period of use of HT was shorter than physicians' recommendations, women's reasons for using HT were usually to treat symptoms, but physicians considered HT also useful in the prevention of later diseases. Gynecologists were more favorable toward HT than other physicians. HT has become common in very different times in different countries, but with the exception of the US experience in the 1970s, the trend has been towards increasing use. One motivation to do surveys on physicians' prescribing or women's use of HT has been to facilitate HT use. The large variation in HT use may reflect the uncertainty concerning its true value. The reasons for the large-scale prevention with HT have not been systematically studied, but it is likely due to various social and commercial forces.     

Breath ethane as a marker of reactive oxygen species during manipulation of diet and oxygen tension in rats

Breath ethane, O2 consumption, and CO2 production were analyzed in 24-mo-old female Fischer 344 rats that had been fed continuously ad libitum (AL) or restricted 30% of AL level (DR) diets since 6 wk of age. Rats were placed in a glass chamber that was first flushed with air, then with a gas mixture containing 12% O2. After equilibration, a sample of the outflow was collected in gas sampling bags for subsequent analyses of ethane and CO2. The O2 and CO2 levels were also directly monitored in the outflow of the chamber. O2 consumption and CO2 production increased for DR rats. Hypoxia decreased O2 consumption and CO2 production for the AL-fed and DR rats. These changes reflect changes in metabolic rate due to diet and PO2. A significant decrease in ethane generation was found in DR rats compared with AL-fed rats. Under normoxic conditions, breath ethane decreased from 2.20 to 1.61 pmol ethane/ml CO2. During hypoxia the levels of ethane generation increased, resulting in a DR-associated decrease in ethane from 2.60 to 1.90 pmol ethane/ml CO2. These results support the hypothesis that DR reduces the level of oxidative stress.     

Induction of spermidine/spermine N1-acetyltransferase in human cancer cells in response to increased production of reactive oxygen species

Hemostatic factors play an important role in the complications of ischemic heart and vessel disease. Dietary fats such as n-3 fatty acids have been shown to possibly influence hemostatic factors. However, most studies reporting an inverse association between cardiovascular disease and fish and n-3 fatty acid consumption used supplemental doses of fish oil or intakes exceeding the typical amount consumed by the US population. This report examined the associations of usual intakes of fish, linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid with fibrinogen, factor VII, factor VIII, and von Willebrand factor in the Coronary Artery Risk Development in Young Adults (CARDIA) Study. The analyses reported here included 1672 black and white men and women aged 24 to 42 years in 1992 to 1993. After adjustment for age, body mass index, diabetes, number of cigarettes smoked per day, race, and energy and alcohol consumption, no significant associations were observed between those who consumed no fish versus those who consumed the highest level of dietary fish with respect to fibrinogen, factor VIII, or von Willebrand factor for any race-sex group. Comparisons of tertile 1 versus tertile 3 for dietary linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid were also not significantly associated with fibrinogen, factor VII, factor VIII, or von Willebrand factor for any race-sex group. These data suggest that customary intakes of fish and n-3 fatty acids in populations that generally do not consume large amounts of these food items are not associated with these hemostatic factors.     

Lipopolysaccharide-induced expression of reactive oxygen species and peroxynitrite in the guinea pig vestibular organ

The purpose of this study was to investigate the occurrence of reactive oxygen species and peroxynitrite in the vestibular organ of the guinea pig following inoculation with bacterial lipopolysaccharide (LPS). The animals were injected transtympanically with 1 mg of LPS 24 h after the intraperitoneal injection of 0.1 mg LPS. Forty-eight hours after the inoculation, varying degrees of degeneration of the vestibular end organs were observed. Immunohistochemical study revealed immunoreactivity to xanthine oxidase (which generates O-2) in the vestibular organ after inoculation with LPS. Immunohistochemical investigation with a specific antinitrotyrosine antibody also showed intense staining of sensory epithelium, fluid transporting cells and the endolymphatic sac, suggesting formation of peroxynitrite in the vestibular organ through the reaction of NO with O-2. On the basis of these data, it can be concluded that NO together with O-2, which form more reactive peroxynitrite, may be the most important pathogenic agents in LPS-induced labyrinthitis in the guinea pig.     

Lead exposure causes generation of reactive oxygen species and functional impairment in rat sperm

The relationships between blood lead, sperm lead, sperm reactive oxygen species (ROS) level, and sperm fertile capability were investigated to understand the effects of lead exposure on sperm function and the mechanism of these effects. Male Sprague-Dawley rats, 7 weeks old, were randomly divided into control group and lead-treated group. The controls and lead-treated animals received intraperitoneal injection of 10 mg sodium acetate and 10 mg lead acetate/kg body weight, respectively, weekly for 6 or 9 weeks. The blood lead and epididymal sperm lead were analyzed by graphite furnace atomic absorption spectrophotometer. Chemiluminescence was measured to evaluate the generation of sperm ROS. Sperm-oocyte penetration rate (SOPR) was measured to evaluate sperm function. After 6 weeks of lead exposure, the rats had average blood lead levels of 32 microg/dl, sperm lead levels of 0.67 +/- 0.11 microg/10(9) sperm, unchanged epididymal sperm counts, percent of motile sperms, and motile epididymal sperm counts compared with control animals. However, after 9 weeks of lead exposure, the rats had average blood lead levels of 48.0 +/- 4.3 microg/dl, sperm lead levels of 0.88 +/- 0.16 microg/10(9) sperm, statistically lower epididymal sperm counts, and lower motile epididymal sperm counts. There was a good correlation between the blood lead and sperm lead(r2 = 0.946, P < 0.001). The sperms of lead-exposed rats produced significantly higher counts ofchemiluminescence than did those from the control rats (P < 0.001). The chemiluminescence counts were positively associated with sperm lead level (r2 = 0.613, P < 0.001). Epididymal sperm counts, motility and motile epididymal sperm counts were negatively associated with sperm chemiluminescence (r2 = 0.255, 0.152, and 0.299; P < 0.01, 0.05, and 0.01, respectively). The SOPR were positively associated with epididymal sperm counts, motility and motile epididymal sperm counts (r2 = 0.136, 0.285, and 0.264; P < 0.05, 0.01, and 0.001, respectively). The sperm chemiluminescence was negatively associated with SOPR (r2 = 0.519, P < 0.001). It is concluded that lead exposure probably affected the sperm function by activating one of the pathways of ROS generation.     

Acute and chronic ethanol increases reactive oxygen species generation and decreases viability in fresh, isolated rat hepatocytes

Although reactive oxygen species (ROS) have been implicated in the etiology of alcohol-induced liver disease, neither their relative contribution to cell death nor the cellular mechanisms mediating their formation are known. The purpose of this study was to test the hypothesis that acute and chronic ethanol exposure enhances the mitochondrial generation of ROS in fresh, isolated hepatocytes. Acute ethanol exposure stimulated ROS production, increased the cellular NADH/NAD+ ratio, and decreased hepatocyte viability slightly, which was prevented by pretreatment with 4-methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase. Similarly, xylitol, an NADH-generating compound, enhanced hepatocyte ROS production and decreased viability. Incubation with pyruvate, an NADH-oxidizing compound, and cyanamide, an inhibitor of aldehyde dehydrogenase, significantly decreased ROS levels in acute ethanol-treated hepatocytes. Chronic ethanol consumption produced a sixfold increase in hepatocyte ROS production compared with levels measured in controls. Hepatocytes from ethanol-fed rats were less viable compared with controls, e.g., viability was 68% +/- 2% (ethanol) versus 83% +/- 1% (control) after 60 minutes of incubation. Antimycin A increased ROS production and decreased cell viability; however, the toxic effect of antimycin A was more pronounced in ethanol-fed hepatocytes. These results suggest that acute and chronic ethanol exposure exacerbates mitochondrial ROS production, contributing to cell death.     

The C1q binding activity of IgG is modified in vitro by reactive oxygen species: implications for rheumatoid arthritis

IgG can be denatured in vitro by reactive oxygen species (ROS). Native IgG activates the complement cascade through C1q. Using a modified ELISA, C1q binding activity of rheumatoid IgG has been compared to IgG denatured by neutrophil-derived ROS. The C1q binding activity of rheumatoid synovial fluid IgG is greater than the corresponding serum IgG (P < 0.01). Denaturation of IgG by activated polymorphs or the Fenton reaction decreased its C1q binding activity (P < 0.01). In vitro exposure of IgG to OH. and ROO. increased its interaction with C1q (P < 0.01). Hypochlorous acid had no effect. ROS-induced alteration to IgG-C1q binding activity may promote the inflammatory response in rheumatoid arthritis.     

Generation of reactive oxygen species and activity of platelet-activating factor acetylhydrolase in human monocyte-derived macrophages

Monocytes were prepared from healthy human volunteers and were allowed to differentiate into macrophages by adhesion to plastic surface and cultured over 7 days in presence of either 10% fetal calf serum (FCS), human control serum or serum from hyperlipaemic patients. Hyperlipaemic serum stimulated the differentiation (measured as an increase in cellular protein and DNA content) to a higher extent when compared to control serum and FCS. With all sera a marked increase of the cellular activity of the enzyme platelet-activating factor acetylhydrolase (PAF-AH) and a tremendous decrease in the capacity of cells to generate reactive oxygen species (ROS) was observed. After seven days of culture the increase in PAH-AH activity was about 19-fold with hyperlipaemic serum, 11-fold with control serum and 6-fold with FCS. During the same period of time ROS generation measured as zymosan-induced chemiluminescence decreased by about 98% and no significant differences between the three types of serum were found. The results indicate that the activity of PAF-AH and the capacity of ROS generation which are both assumed to play an important role in the oxidation of low-density lipoproteins (LDL) and thus in the development of atherosclerosis, change in opposite direction during the differentiation of blood monocytes into macrophages, and that hyperlipaemic serum stimulates PAF-AH activity but not ROS generation.