The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium

In order to clarify the features of reactive oxygen species produced by rat alveolar macrophages (AMs), the concentrations of intracellular and extracellular hydrogen peroxide were measured under various experimental conditions. Intracellular hydrogen peroxide was measured by DCFH method using a flow cytometer, while the extracellularly released fraction was measured by scopoletin method using a spectrophotometer. The concentration of intracellular hydrogen peroxide after stimulation with opsonized zymosan (10 micrograms/ml) was significantly higher than that after stimulation with phorbol myristate acetate (PMA; 100 ng/ml). On the other hand, hydrogen peroxide released extracellularly after stimulation with PMA was significantly greater that that after stimulation with opsonized zymosan. These results indicate that the soluble membrane stimulant and the phagocytic particles have different mechanisms in activating the production of hydrogen peroxide in AMs. That is, hydrogen peroxide induced by PMA was mainly released extracellularly, while that induced by zymosan was mainly released into the intracellular environment. At rest, the concentration of intracellular hydrogen peroxide in rat AMs was high. Potassium cyanate, a known mitochondrial inhibitor, suppressed the intracellular hydrogen peroxide in AMs not only at rest but also after stimulations, indicating that most of the reactive oxygen species released into the intracellular environment in AM are produced by mitochondria. From these results, in order to gain a closer insight into the function of AMs, it is very important to distinguish the oxidative metabolites produced intracellularly which are related to bactericidal function from those of the extracellularly released fraction which give rise to lung damage.

We have previously shown that alpha-melanocyte-stimulating hormone (alpha-MSH) can oppose tumor necrosis factor alpha activation of NF-kappaB (1-2 h) and intercellular adhesion molecule 1 up-regulation (mRNA by 3 h and protein by 24 h) in melanocytes and melanoma cells. The present study reports on the ability of four MSH peptides to control intracellular peroxide levels and glutathione peroxidase (GPx) activity in pigmentary and nonpigmentary cells. In human HBL melanoma and HaCaT keratinocytes tumor necrosis factor alpha and H(2)O(2) both activated GPx in a time- and concentration-dependent manner (by 30-45 min). alpha-MSH peptides were found to inhibit the stimulated GPx activity and had biphasic dose-response curves. MSH 1-13 and MSH [Nle(4)-d-Phe(7)] achieved maximum inhibition at 10(-10) and 10(-12) m, respectively. Higher concentrations (10-100 fold) of MSH 4-10 and MSH 11-13 were required to produce equivalent levels of inhibition. alpha-MSH was also capable of reducing peroxide accumulation within 15 min, and again this inhibition was biphasic. The data support a role of alpha-MSH in acute protection of cells to oxidative/cytokine action that precedes NF-kappaB and GPx activation. The rapidity and potency of the response to alpha-MSH in pigmentary and nonpigmentary cells suggest this to be a central role of this peptide in cutaneous cells.

Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm

To clarify the mechanisms of intracellular induction of oxidative DNA damage, we have investigated the concentrations of intracellular reactive oxygen species and the amounts of 8-hydroxydeoxyguanosine (8OHdG), a mutagenic oxidative DNA damage, in human neutrophil-like cells, dimethylsulfoxide-differentiated HL60 (DMSO-HL60). We determined intracellular concentrations of hydrogen peroxide and superoxide by flow cytometry with dichlorofluorescein diacetate and hydroethidine, respectively. We determined the 8OHdG amounts with an electrochemical detector connected to HPLC after anaerobic sample processing. DMSO-HL60 releases superoxide upon stimulation with phorbol myristate acetate, and the released superoxide dismutates to hydrogen peroxide. Stimulation of DMSO-HL60 with 100 nM phorbol myristate acetate increased intracellular hydrogen peroxide, superoxide and 8OHdG (control). Addition of 1000 U/ml catalase decreased hydrogen peroxide (31.3% of control) and 8OHdG (20.3%). Addition of 100 U/ml SOD decreased superoxide (18.7%) and 8OHdG (41.6%). Addition of 1 mM deferoxamine decreased 8OHdG (30.4%), but increased hydrogen peroxide (129.6%). Addition of 200 microM 4-acetamido-4'- isothiocyanostilbene-2,2'-disulfonic acid decreased superoxide (59.9%) and 8OHdG (42.0%). Addition of 0.4% ethanol had no effect on superoxide concentration (102.2%), but tended to decrease hydrogen peroxide (83.5%) and 8OHdG (84.3%). Pretreatment of DMSO-HL60 with 0.1 mM FeSO4 increased 8OHdG (117.3%), but decreased hydrogen peroxide (75.8%). These findings indicate that the extracellularly released superoxide and hydrogen peroxide diffuse into the cell, but that such reactive oxygen species are not the direct molecules to induce 8OHdG. Our results suggest that 8OHdG is induced by the hydroxyl radical which is generated from intracellular hydrogen peroxide and superoxide-reduced Fe.

Molecular hydrogen (H2) is recognized as a gaseous antioxidant, and it is expected to ameliorate various disorders related to oxidative stress and inflammation. However, there are still many unclear points regarding its effectiveness in the skin. Therefore, the purpose of this study was to examine the protective effect of H2 against ultraviolet (UV) irradiation-related stress injury in human epidermal HaCaT cells. We investigated the effects of H2 against three types of UV-derived oxidative stress using human skin keratinocytes: hydrogen peroxide (H2O2)-induced oxidative stress, tert-butyl hydroperoxide (t-BuOOH)-induced lipid peroxidation stress, and glyoxal-induced carbonyl stress. Our results showed that H2 exerted cytoprotective effects against stress induced by H2O2, t-BuOOH, and glyoxal. Furthermore, our results also revealed that H2 suppressed H2O2-induced increases in intracellular peroxide and H2O2 levels, and suppressed the progression of lipid peroxidation. Taken together, our results demonstrate that H2 can exert protective effects against oxidative stress-, lipid peroxidation-, and carbonyl stress-induced cellular injuries in human keratinocytes, partly mediated via suppression of intracellular oxidative stress and peroxide generation. Therefore, H2 is expected to be utilized as an effective and attractive component in cosmetic formulations in the future.

Detection of intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) and lipid peroxidation during cryopreservation of alpaca spermatozoa.

Doxorubicin increases intracellular hydrogen peroxide in PC3 prostate cancer cells

Real-time bacterial detection with an intracellular ROS sensing platform

DNA is continuously degraded by numerous genotoxic agents including intracellular reactive oxygen species produced by cell metabolism and exogenous environmental pollutants. These genotoxic agents destroy the DNA integrity leading to carcinogenesis or cell death. An increased consumption of vegetables, fruits and other foods rich in antioxidant compounds can protect DNA from oxidative damage and prevent cell carcinogenesis. This study was designed to investigate in vitro the genoprotective and DNA repair activities of the fruit pulp ethanol extract from Detarium microcarpum against two known mutagenic agents such as hydrogen peroxide and methyl methane sulfonate. To assess the genoprotective and DNA repair activities of extract, human lymphocytes in culture were treated with the extract before or after the genotoxic agent exposing. The amount of DNA damages was assessed by using the standard comet assay. The fruit pulp extract in concentration up to 500 µg/mL, compared to vehicle didn’t affect the integrity of DNA. Interestingly, the genotoxic effects of hydrogen peroxide and methyl methane sulfonate on human lymphocytes were significantly reduced by the extract pre-treatment. In addition, the DNA damages induced by hydrogen peroxide and methyl methane sulfonate were repaired further to the extract addition. The fruit pulp ethanol extract from Detarium microcarpum contains bioactive compounds that can preserve the integrity of DNA from the deleterious effects of genotoxic agents. The daily intake of this fruit pulp as food supplement could prevent DNA damages and carcinogenesis.

Effect of FertTalp addition to stallion semen on kinetic parameters and production of intracellular hydrogen peroxide after thawing

When DNA is introduced into eukaryotic cells, it can be integrated into the genome by homologous or illegitimate recombination 1,2. Despite great efforts to gain insight into the molecular mechanisms, our understanding of the recombination process is still in its infancy. In the absence of a molecular model, predictions concerning the frequency of homologous recombination compared to illegitimate recombination cannot be precisely made. In mammalian cells, illegitimate recombination is the most predominant event (for review, seeref. (3). Thus, if the integration of DNA via homologous recombination into mammalian cells is the goal of the experiment, a single homologous recombination event has to be detected among many illegitimate recombination events. Described here is a method of detecting homologous recombination events in a small subpopulation of cells by using the polymerase chain reaction (PCR), a primer-directed enzymatic amplification of specific DNA sequences. This method can be used to determine the homologous recombination frequency and to facilitate the cloning of homologously recombined cells 1,2. Homologously recombined alleles are identified by their amplification products, which are generated by the PCR reaction (see Fig. 1). The specificity of the reaction is dependen t on the two primers. One primer (primer 2 in Fig. 1) primes DNA synthesis specifically at the nonhomologous sequences of the exogenous DNA. The other primer (primer 1 in Fig. 1) is specific for the target locus, but outside of the exogenous DNA. Thus, both priming sites are physically linked in a predictable manner only after homologous recombination. Fig. 1. Detection of homologous recombination events by PCR. Homologous recombination between a target locus and exogenous DNA yields a new recombinant molecule in which a small portion of the target locus is replaced by a heterologous sequence of the exogenous DNA. This recombination event links two priming sites for the PCR. Primer 1 primes DNA synthesis from the target sequence, outside of the region homologous to the exogenous DNA. Primer 2 is specific for the heterologous sequence residing in the exogenous DNA. These priming sites are not linked (in the same manner) after the integration of the exogenous DNA by illegitimate recombination. Yet, DNAs of a heterogeneous length are made from the target and the illegitimately recombined sequence during every PCR cycle. Those molecules that terminate in the region of homology could anneal, thus creating a recombinant molecule.

Cryopreservation impairs sperm quality and functions, including motility and DNA integrity. Antioxidant additives in sperm freezing media have previously brought improvements in postthawed sperm quality. Green tea extract (GTE) is widely considered as an excellent antioxidant, and its beneficial role has been proven in other human cells. This study aims to evaluate the GTE as a potential additive in cryopreservation media of human spermatozoa. In part one, the semen of 20 normozoospermic men was used to optimize the concentration of GTE that maintains sperm motility and DNA integrity against oxidative stress, induced by hydrogen peroxide (H2O2). Spermatozoa were treated with GTE at different concentrations before incubation with H2O2. In part two, the semen of 45 patients was cryopreserved with or without 1.0 ng ml−1 GTE. After 2 weeks, the semen was thawed, and the effect on sperm motility and DNA fragmentation was observed. Our data showed that GTE significantly protected sperm motility and DNA integrity against oxidative stress induced by H2O2 when added at a final concentration of 1.0 ng ml−1. We found that the addition of 1.0 ng ml−1 GTE to cryopreservation media significantly increased sperm motility and DNA integrity (both P < 0.05). More interestingly, patients with high sperm DNA damage benefited similarly from the GTE supplementation. However, there was no significant change in the reactive oxygen species (ROS) level. In conclusion, supplementing sperm freezing media with GTE has a significant protective effect on human sperm motility and DNA integrity, which may be of clinical interest.

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

Post-thaw addition of pentoxifylline to equine semen previously cryopreserved with different concentrations of cholesterol-loaded cyclodextrin

Hydrogen peroxide is a toxic metabolite of oxidative metabolism, but may also serve as a broad spectrum signaling molecule controlling a variety of essential physiological processes. Photoperiod influences diapause beyond the maternal generation in Cotesia vestalis and we speculated that intracellular hydrogen peroxide was a possible signal mediating this generational effect. To investigate the signaling functions of hydrogen peroxide in diapause induction in C. vestalis, hydrogen peroxide and activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase were measured at diapause, and in maternal non-diapausing prepupa, adult, and offspring egg stages form the cultures reared at 25°C under the photoperiods of 8∶16, 12∶12, and 16∶8 (L∶D) h. The hydrogen peroxide contents and activities of superoxide dismutase and peroxidase in maternal non-diapausing prepupae and adults declined as the light period was reduced, whereas catalase activity increased significantly. Compared to d...