Biochemical changes associated with in vivo RbcL fragmentation by reactive oxygen species under chilling-light conditions

During physiological stress, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) degradation is accelerated, which is considered to be one of the key factors responsible for photosynthetic decline. A recent study has shown that the large subunit (RbcL) of Rubisco is directly fragmented by hydroxyl radicals in Cucumis sativus leaves under chilling-light conditions. In the present study, we investigated biochemical aspects associated with this in vivo RbcL fragmentation by reactive oxygen species. RbcL fragmentation was observed in C. sativus and Phaseolus vulgaris , but not in Solanum lycopersicum , Glycine max , Oryza sativa , Triticum aestivum , Spinacia oleracea or Arabidopsis thaliana . In C. sativus and P. vulgaris , RbcL fragmentation followed the fragmentation of PsaB, while in the other species, PsaB fragmentation did not occur. In C. sativus and P. vulgaris , the activities of antioxidant enzymes decreased dramatically under chilling-light conditions, and the proportion of uncarbamylated Rubisco increased. These data suggest that in vivo RbcL fragmentation under chilling-light conditions is associated with a combination of events, namely, inactivation of antioxidant enzymes, destruction of photosystem I and an increase of uncarbamylated Rubisco, which can produce hydroxyl radicals via the Fenton reaction at the catalytic site of RbcL.     

Bactericidal activity of Ag-zeolite mediated by reactive oxygen species under aerated conditions

The bactericidal activity induced by the introduction of silver ions into zeolite was studied. Escherichia coli was used as the test microorganism. Silver ions were loaded into zeolite by the ion-exchange method. Silver-loaded zeolite was demonstrated the strong bactericidal activity. Dissolved oxygen was an essential factor for the occurrence of the bactericidal activity because the activity was observed only under aerated condition. Superoxide anions, hydrogen peroxide, hydroxyl radicals and singlet oxygen were formed. Scavengers of these each reactive oxygen species (ROS) inhibited the bactericidal activity. This means that all ROS contributed to the activity.     

The oxygen sensing signal cascade under the influence of reactive oxygen species

Structural and functional integrity of organ function profoundly depends on a regular oxygen and glucose supply. Any disturbance of this supply becomes life threatening and may result in severe loss of organ function. Particular reductions in oxygen availability (hypoxia) caused by respiratory or blood circulation irregularities cannot be tolerated for longer periods due to an insufficient energy supply by anaerobic glycolysis. Complex cellular oxygen sensing systems have evolved to tightly regulate oxygen homeostasis. In response to variations in oxygen partial pressure (PO2), these systems induce adaptive and protective mechanisms to avoid or at least minimize tissue damage. These various responses might be based on a range of oxygen sensing signal cascades including an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial chain such as a specialized mitochondrial, low PO2 affinity cytochrome c oxidase (aa3) and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF (hypoxia inducible factor) prolyl-hydroxylase and HIF asparaginyl hydroxylase called factor-inhibiting HIF (FIH-1). Thus, specific oxygen sensing cascades involving reactive oxygen species as second messengers may by means of their different oxygen sensitivities, cell-specific and subcellular localization help to tailor various adaptive responses according to differences in tissue oxygen availability.     

Performance of two Rhinanthus species under different hydric conditions

Rhinanthus angustifolius and Rhinanthus minor are annual hemiparasitic herbs found in open vegetations. They are closely related, hybridize frequently, and appear to have largely overlapping niches, although some floras report a preference of R. angustifolius for more humid conditions compared to R. minor. We analysed the relative fitness of both species by following their fate from germination to seed production in three different hydric treatments (wet, moist and dry) in a garden experiment. We found that R. angustifolius was either as fit as or fitter than R. minor in all conditions and for all studied parameters, with a slightly higher flower production in R. angustifolius in the wet treatment. Wet conditions had a negative effect on germination and flowering rate, but they increased flower and seed production. These observations show that environmental conditions will influence the composition of mixed Rhinanthus populations.     

Origin of reactive oxygen species in human semen: spermatozoa or leucocytes?

Peroxidative damage induced by reactive oxygen species (ROS) has been proposed as one of the major causes of defective sperm function. In previous studies of the production of ROS in semen, the contribution of contaminating leucocytes was not assessed. We determined the levels of ROS in 60 semen samples from men attending our infertility clinic and demonstrated by performing extraction experiments with antibody-coated magnetic beads that, within this unselected population of patients, leucocytes were the major source of ROS in the low-density Percoll fraction. Of the sperm motion parameters examined using computerized semen analysis, beat-cross frequency was the only one significantly affected by the ROS in semen.     

ATM at the crossroads of reactive oxygen species and autophagy

Reactive oxygen species (ROS) are generally small, short-lived and highly reactive molecules, initially thought to be a pathological role in the cell. A growing amount of evidence in recent years argues for ROS functioning as a signaling intermediate to facilitate cellular adaptation in response to pathophysiological stress through the regulation of autophagy. Autophagy is an essential cellular process that plays a crucial role in recycling cellular components and damaged organelles to eliminate sources of ROS in response to various stress conditions. A large number of studies have shown that DNA damage response (DDR) transducer ataxia-telangiectasia mutated (ATM) protein can also be activated by ROS, and its downstream signaling pathway is involved in autophagy regulation. This review aims at providing novel insight into the regulatory mechanism of ATM activated by ROS and its molecular basis for inducing autophagy, and revealing a new function that ATM can not only maintain genome homeostasis in the nucleus, but also as a ROS sensor trigger autophagy to maintain cellular homeostasis in the cytoplasm.     

Molecular mechanisms of cellular injury produced by neurotoxic amino acids that generate reactive oxygen species

Summary There is now strong experimental evidence that the basic precursors for the synthesis of catechol(amine) and indolamine neurotransmitters, tyrosine and tryptophan can act as generators of ROS (reactive oxygen species): peroxides, superoxide and peroxyradicals. The consequences of free radicals formation from precursors during oxidative degradation process, their possible participation in electron transfer/addition reactions and chain processes involving cell antioxidant defense system were presented and discussed. Although the generation of neurotoxic ROS by tyrosine and tryptophan is accepted to occur in the presented model systems, doubts can exist as to the situationin vivo, which may be completely different and remain to be explored. The relevance of the present findings with regard to a variety of neurological diseases cannot be ignored.     

Effect of oxygen availability on nitrogen fixation by two Lotus species under flooded conditions

The pasture legumes Lotus uliginosus (Schk.) and Lotus corniculatus (L.), known to differ in their tolerance to flooding, were inoculated with Rhizobium loti and flooded for 60 d while subjected to two levels of dissolved pO2: 0.241 and 0.094 mol ml^-1. L. uliginosus showed significantly greater growth (shoot and root) and N2 fixation under both pO2s, compared to L. corniculatus, although growth and N2 fixation by L. corniculatus was not affected by the low pO2. Surprisingly, in L. uliginosus., growth, nodulation and N2 fixation were all increased by low pO2 while nodulation of L. corniculatus where low pO2 plants showed a significant increase over that of the higher pO2 plants while L. uliginosus plants showed a decline. Root porosity of L. uliginosus doubled in the low pO2-treatment from a mean of 14.5% in high pO2 roots to 28.5%, whereas that of L. corniculatus was relatively unaffected by pO2, being 7% and 9% for high and low pO2 plants, respectively. The structure of nodules differed little between species and treatments, although nodules/nodulated roots from the L. uliginosus plants had particularly profuse lenticels and aerenchyma. However, L. corniculatus nodules, especially those grown in the lower pO2 showed signs of early senescence with vacuolation of infected cells and green coloration when cut open. Leghaemoglobin (Lb) concentrations in nodules from both species were unaffected by low pO2, although that of L. corniculatus nodules, regardless of pO2, was significantly greater than L. uliginosus. Concentrations of the intercellular glycoprotein recognized by the monoclonal antibody MAC265 were significantly reduced in nodules from the low pO2 treatment in both species. Immunogold labelling showed that the MAC265 antigen was localized primarily within intercellular spaces within nodule cortices from both Lotus species. A marked decrease in deposition of the MAC265 antigen within the cortices of L. uliginosus nodules grown in the lower pO2, is discussed in terms of the relative abilities of the two Lotus spp. to maintain an O2 supply to the N2-fixing bacteroids within submerged nodules.Keywords: Lotus uliginosus, Lotus corniculatus, N2 fixation, flooding, oxygen.      

Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing

In the present study, we provide evidence for the production of reactive oxygen species (ROS) during cryopreservation of bovine spermatozoa. Cooling and thawing of spermatozoa cause an increase in the generation of superoxide radicals. Although nitric oxide production remains unaltered during sperm cooling from 22-4 degrees C, a sudden burst of nitric oxide radicals is observed during thawing. Increase in lipid peroxidation levels have been observed in frozen/thawed spermatozoa and appears to be associated with a reduction in sperm membrane fluidity as detected by spin labeling studies. The data presented provide strong evidence that oxygen free radicals are produced during freezing and thawing of bovine spermatozoa and suggest that these reactive oxygen species may be a cause for the decrease in sperm function following cryopreservation. Mol. Reprod. Dev. 59: 451-458, 2001.     

Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.     

Generation of reactive oxygen species from Hinokitiol under near-UV irradiation

Near-UV irradiation caused the decomposition of hinokitiol in an aqueous solution. During the photochemical reaction, the distinct electron spin resonance signal characteristic of the adduct of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) with the hydroxyl radical was accompanied by small signals corresponding to the adduct of DMPO with the superoxide anion radical. More than 95% of Escherichia coli cells were killed by the incubation with hinokitiol under near-UV irradiation by BLB fluorescent lamps. These results indicated the generation of reactive oxygen species during photochemical reaction of hinokitiol under near-UV irradiation.     

Impact of precise modulation of reactive oxygen species levels on spermatozoa proteins in infertile men

Background

Elevated levels of reactive oxygen species (ROS) are detected in 25% to 80% of infertile men. They are involved in the pathology of male infertility. Understanding the effect of increasing levels of ROS on the differential expression of sperm proteins is important to understand the cellular processes and or/pathways that may be implicated in male infertility. The aim of this study was to examine differentially expressed proteins (DEPs) in spermatozoa from patients with low, medium and high ROS levels.

Methods

A total of 42 infertile men presenting for infertility and 17 proven fertile men were enrolled in the study. ROS levels were measured by chemiluminescence assay. Infertile men were divided into Low (0- < 93 RLU/s/10⁶ sperm) (n = 11), Medium (>93-500 RLU/s/10⁶ sperm) (n = 17) and High ROS (>500 RLU/s/10⁶ sperm) group (n = 14). All fertile men had ROS levels between 4-50 RLU/s/10⁶ sperm. 4 subjects from fertile group and 4 each from the Low, Medium and High ROS were pooled. Protein extraction, protein estimation, gel separation of the proteins, in-gel digestion, LTQ-orbitrap elite hybrid mass spectrometry system was conducted. The DEPs, the cellular localization and pathways of DEPs involved were examined utilizing bioinformatics tools.

Results

1035 proteins were identified in the 3 groups by global proteomic analysis. Of these, 305 were DEPs. 51 were unique to the Low ROS group, 47 Medium ROS group and 104 were unique to the High ROS group. 6 DEPs were identified by Uniprot and DAVID that had distinct reproductive functions and they were expressed only in 3 ROS groups but not in the control.

Conclusions

We have for the first time demonstrated the presence of 6 DEPs with distinct reproductive functions only in men with low, medium or high ROS levels. These DEPs can serve as potential biomarkers of oxidative stress induced male infertility.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-12-4) contains supplementary material, which is available to authorized users.     

Effect of antioxidant supplementation on semen quality and reactive oxygen species of frozen-thawed canine spermatozoa

The objective of this study was to evaluate post-thaw quality of frozen dog semen processed with diluents containing different antioxidants. Ejaculates were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF movement), viability, acrosomal integrity and by the hypo-osmotic swelling test. Also, superoxide production, hydroxyl radicals and total reactive oxygen species (tROS) were determined. The pool was divided in seven aliquots, for control and test conditions, which were processed for cryopreservation. The sperm pellets were diluted to a final concentration of 200x10(6)sperm/ml with TRIS-glucose-egg yolk extender containing one of the following supplements: vitamin C (1.5mM), NAC (N-acetyl-l-cysteine; 1.5mM), taurine (0.6mM), catalase (300U/ml), vitamin E (0.3mM) and B16 [5-(4-dimethylamino-phenyl)-2-phenyl-penta-2,4-dienoic acid; 0.3mM]. Post-thaw semen evaluation showed that mean (+/-S.E.M.) motility was increased (p<0.001) after addition of catalase (49.75+/-3.63 versus 39.00+/-2.90 in controls), whereas more spermatozoa with RSF movement were observed (p<0.001) after the catalase, NAC and vitamin E treatments (31.75+/-3.46, 28.00+/-3.27, 26.75+/-3.15, respectively, versus 17.00+/-2.26 in controls). Viability was increased (p<0.001) after addition of catalase, taurine, NAC and tocopherol (66.00+/-3.03, 61.90+/-2.48, 60.60+/-1.93 and 60.50+/-4.12, respectively, versus 51.70+/-2.81 in controls). The percentage of swollen spermatozoa was increased after addition of catalase and taurine (61.75+/-1.61 and 61.25+/-1.49, respectively, versus 55.65+/-1.64 in controls). Acrosomal integrity was not influenced in any case. B16 addition had adverse effects on all parameters evaluated. None of the reactive oxygen species were significantly reduced post-thaw in antioxidant treated semen. The results suggest that catalase had the most pronounced effect in improving post-thaw quality of canine spermatozoa.     

Generation of reactive oxygen species in water under exposure of visible or infrared irradiation at absorption band of molecular oxygen

It is found that in bidistilled water saturated with oxygen hydrogen peroxide and hydroxyl radicals are formed under the influence of visible and infrared radiation in the absorption bands of molecular oxygen. Formation of reactive oxygen species (ROS) occurs under the influence of both solar and artificial light sourses, including the coherent laser irradiation. The oxygen effect, i.e. the impact of dissolved oxygen concentration on production of hydrogen peroxide induced by light, is detected. It is shown that the visible and infrared radiation in the absorption bands of molecular oxygen leads to the formation of 8-oxoguanine in DNA in vitro. Physicochemical mechanisms of ROS formation in water when exposed to visible and infrared light are studied, and the involvement of singlet oxygen and superoxide anion radicals in this process is shown.     

Generation of reactive oxygen species by equine neutrophils and their effect on motility of equine spermatozoa

Contaminating leukocytes in the ejaculate are an important source of reactive oxygen species (ROS) in human semen. When present in sufficient numbers, they can have a detrimental influence on sperm function in humans. Unfortunately, there is little published information regarding the importance of leukocytes in stallion semen. The objectives of this study were to determine the production of hydrogen peroxide (H2O2) by activated equine neutrophils and to examine the effect of this ROS production on equine sperm motility in vitro. Motile equine spermatozoa (two ejaculates each from four stallions) and peripheral blood neutrophils were isolated on discontinuous Percoll gradients, washed and resuspended in a modified Tyrode's medium. Spermatozoa (25 x 10(6)/ml) were incubated for 30 min at 38 C with neutrophils (0,0.5 x 10(6),1 x 10(6), 5 x 10(6) and 10 x 10(6)/ml) activated by either the protein kinase C agonist, 12-myristate, 13-acetate phorbol ester (PMA; 100 nM) or the leukocyte chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP; 0.1 mM). Sperm motility was determined by computer-assisted semen analysis (CASA) at time 0 min (T0) and time 30 min (T30), and H2O2 was measured at T30 with the Amplex Red assay kit. At T30, there was a significant (P < 0.01) increase in H2O2 with the addition of 5 x 10 and 10 x 10(6) neutrophils/ml activated by FMLP (0.76 +/- 0.3 and 0.99 +/- 0.4 microM, respectively, versus 0.0024 +/- 0.002 microM in sperm alone), and this increase was associated with a significant (P < 0.001) decrease in total motility (52 +/- 5.1 and 48 +/- 6.0%, respectively, versus 80 +/- 4.7% in sperm alone). At T30, there was also a significant (P < 0.001) increase in H2O2 with the addition of 5 x 10(6) and 10 x 10(6) neutrophils/ml activated by PMA (1.88 +/- 0.2 and 2.07 +/- 0.3 microM, respectively, versus 0.0009 +/- 0.0006 microM in sperm alone). The results of this study demonstrate that 5 x 10(6) activated neutrophils/ml are sufficient to impair equine sperm motility in vitro.