Increased Peroxide Generation Research Articles

The synchronicity of bloom-forming cyanobacteria transcription patterns and hydrogen peroxide dynamics

Hydrogen peroxide is a reactive oxygen species (ROS) naturally occurring at low levels in aquatic environments and production varies widely across different ecosystems. Oxygenic photosynthesis generates hydrogen peroxide as a byproduct, of which some portion can be released to ambient water. However, few studies have examined hydrogen peroxide dynamics in relation to cyanobacterial harmful algal blooms (cHABs). A year-long investigation of algal succession and hydrogen peroxide dynamics was conducted at the Caloosahatchee River, Florida, USA. We aimed to identify potential biological mechanisms responsible for elevated hydrogen peroxide production during cHAB events through the exploration of the freshwater microbial metatranscriptome. Hydrogen peroxide concentrations were elevated from February to September of 2021 when cyanobacteria were active and abundant. We observed one Microcystis cHAB event in spring and one in winter. Both had distinct nutrient uptake and cyanotoxin gene expression patterns. While meaningful levels of microcystin were only detected during periods of elevated hydrogen peroxide, cyanopeptolin was by far the most expressed cyanotoxin during the spring bloom when hydrogen peroxide was at its yearly maxima. Gene expressions of five microbial enzymes (Rubisco, superoxide dismutase, cytochrome b559, pyruvate oxidase, and NADH dehydrogenase) positively correlated to hydrogen peroxide concentrations. Additionally, there was higher nitrogen-fixing gene (nifDKH) expression by filamentous cyanobacteria after the spring bloom but no secondary bloom formation occurred. Overall, elevated environmental hydrogen peroxide concentrations were linked to cyanobacterial dominance and greater expression of specific enzymes in the photosynthesis of cyanobacteria. This implicates cyanobacterial photosynthesis and growth results in increased hydrogen peroxide generation as reflected in measured environmental concentrations.

Distinct Redox Signalling following Macrophage Activation Influences Profibrotic Activity

Aims To date, the ROS-generating capacities of macrophages in different activation states have not been thoroughly compared. This study is aimed at determining the nature and levels of ROS generated following stimulation with common activators of M1 and M2 macrophages and investigating the potential for this to impact fibrosis. Results Human primary and THP-1 macrophages were treated with IFN-γ+LPS or IL-4-activating stimuli, and mRNA expression of established M1 (CXCL11, CCR7, IL-1β) and M2 (MRC-1, CCL18, CCL22) markers was used to confirm activation. Superoxide generation was assessed by L-012-enhanced chemiluminescence and was increased in both M(IFN-γ+LPS) and M(IL-4) macrophages, as compared to unpolarised macrophages (MΦ). This signal was attenuated with NOX2 siRNA. Increased expression of the p47phox and p67phox subunits of the NOX2 oxidase complex was evident in M(IFN-γ+LPS) and M(IL-4) macrophages, respectively. Amplex Red and DCF fluorescence assays detected increased hydrogen peroxide generation following stimulation with IL-4, but not IFN-γ+LPS. Coculture with human aortic adventitial fibroblasts revealed that M(IL-4), but not M(IFN-γ+LPS), enhanced fibroblast collagen 1 protein expression. Macrophage pretreatment with the hydrogen peroxide scavenger, PEG-catalase, attenuated this effect. Conclusion We show that superoxide generation is not only enhanced with stimuli associated with M1 macrophage activation but also with the M2 stimulus IL-4. Macrophages activated with IL-4 also exhibited enhanced hydrogen peroxide generation which in turn increased aortic fibroblast collagen production. Thus, M2 macrophage-derived ROS is identified as a potentially important contributor to aortic fibrosis.

Redox responses in skeletal muscle following denervation

Previous studies have shown a significant increase in the mitochondrial generation of hydrogen peroxide (H2O2) and other peroxides in recently denervated muscle fibers. The mechanisms for generation of these peroxides and how the muscle responds to these peroxides are not fully established. The aim of this work was to determine the effect of denervation on the muscle content of proteins that may contribute to mitochondrial peroxide release and the muscle responses to this generation. Denervation of the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles in mice was achieved by surgical removal of a small section of the peroneal nerve prior to its entry into the muscle. An increase in mitochondrial peroxide generation has been observed from 7 days and sustained up to 21 days following denervation in the TA muscle fibers. This increased peroxide generation was reduced by incubation of skinned fibers with inhibitors of monoamine oxidases, NADPH oxidases or phospholipase A2 enzymes and the muscle content of these enzymes together with peroxiredoxin 6 were increased following denervation. Denervated muscle also showed significant adaptations in the content of several enzymes involved in the protection of cells against oxidative damage. Morphological analyses indicated a progressive significant loss of muscle mass in the TA muscle from 7 days up to 21 days following denervation due to fiber atrophy but without fiber loss. These results support the possibility that, at least initially, the increase in peroxide production may stimulate adaptations in an attempt to protect the muscle fibers, but that these processes are insufficient and the increased peroxide generation over the longer term may activate degenerative and atrophic processes in the denervated muscle fibers.

Structure-toxicity relationships of saturated and unsaturated free fatty acids for elucidating the lipotoxic effects in human EndoC-βH1 beta-cells

Lipotoxicity has been considered a major cause for beta-cell dysfunction in type 2 diabetes mellitus. However, the underlying mechanisms are still unclear. To achieve a better understanding of the toxicity a wide range of structurally different free fatty acids (FFAs) has been analyzed in human EndoC-βH1 beta-cells.Exposure of human EndoC-βH1 beta-cells to physiological saturated and monounsaturated long-chain FFAs induced apoptosis. Particularly noteworthy was that the toxicity increased more rapidly with increasing chain length of saturated than of unsaturated FFAs. The highest toxicity was observed in the presence of very long-chain FFAs (C20-C22), whereas polyunsaturated FFAs were not toxic. Long-chain FFAs increased peroxisomal hydrogen peroxide generation slightly, while very long-chain FFAs increased hydrogen peroxide generation more potently in both peroxisomes and mitochondria. The greater toxicity of very long-chain FFAs was accompanied by hydroxyl radical formation, along with cardiolipin peroxidation and ATP depletion. Intriguingly, only saturated very long-chain FFAs activated ER stress. On the other hand saturated very long-chain FFAs did not induce lipid droplet formation in contrast to long-chain FFAs and unsaturated very long-chain FFAs.The present data highlight the importance of structure-activity relationship analyses for the understanding of the mechanisms of lipotoxicity. Chain length and degree of saturation of FFAs are crucial factors for the toxicity of FFAs, with peroxisomal, mitochondrial, and ER stress representing the major pathogenic factors for induction of lipotoxicity. The results might provide a guide for the composition of a healthy beta-cell protective diet.

OP-21 - Redox responses to denervation in skeletal muscle

Previous studies have shown increased mitochondrial generation of peroxides in denervated muscle fibers, but the role that peroxides play during denervation is still controversial. The Anterior Tibialis and Extensor Digitorum Longus muscles of wild type and Thy-1YFP mice were denervated by surgical removal of a small section of the peroneal nerve. Western blotting and immunohistochemical techniques were used to examine protein contents along with analysis of morphological changes and mitochondrial peroxide release following denervation. Data indicate that increased peroxide generation is maintained up to 21 days following denervation and is associated with a significant increase in the muscle contents of Peroxiredoxin 6 and Phospholipase A2 involved in the activation of NADPH Oxidase. A significant increase in the contents of several antioxidant enzymes and HSPs involved in the protection against oxidative damage was also observed. These results support the possibility that (at least initially) an increase in mitochondrial peroxide production may stimulate adaptation and repair processes in the neuromuscular system but sustained increased peroxide generation over the longer term lead to degeneration and muscle atrophy.

EFFECT OF DENERVATION ON REDOX‐REGULATION IN SKELETAL MUSCLE

Changes in skeletal muscle mass and function occur during aging and are accompanied by loss of integrity and stability of the neuromuscular junction. Previous studies from our group have shown a significant increase in the mitochondrial generation of hydrogen peroxide (H202) and other peroxides in denervated muscle fibers. The mechanisms of generation of these peroxides and the effect on muscle responses are unknown. Our aim was to determine the effect of denervation on proteins that may contribute to peroxide release and proteins that regulate redox responses.The Anterior Tibialis (AT) and Extensor Digitorum Longus (EDL) muscles of wild type mice were denervated under anaesthesia by surgical removal of a small section of the peroneal nerve. Western blotting and immunohistochemical techniques were used to examine protein contents. AT and EDL muscles of thy‐1YFP mice, in which YFP expression in neuronal cells allow visualisation of the muscle innervation, were also denervated and used for morphological examination and analysis of muscle mitochondrial peroxide release.Previous work from our group has shown that, following denervation, hydrogen peroxide can be generated by several sources within mitochondria (Pollock et al, Free Rad. Biol. Med 2017) and the current data show that the increased peroxide generation was maintained to 21 days and was associated with a significant increase in Monoamine Oxidase A and NADPH Oxidase components. An increased content of proteins involved in the activation of the NADPH oxidase complex such as Peroxiredoxin 6 and Phospholipase A2 was also observed together with a significant increase in the content of several enzymes involved in the protection of cells against oxidative damage. Oxyblot analysis to assess the levels of carbonylation, showed that by 7 days post‐denervation there was a significant increase in protein carbonylation of low molecular weight (25–35 kDa) proteins. Morphological analysis indicated a progressive significant loss of muscle mass in the AT muscle from 7 days up to 21 days following denervation due to fiber atrophy without fiber loss. These results support the possibility that (at least initially) the increase in peroxide production may stimulate adaptations to protect the muscle fibers but, together with data on structural changes indicating significant fiber atrophy from 7 days after denervation, increased peroxide generation over the longer term may activate degenerative processes, such as apoptosis of the denervated muscle fibers.Support or Funding InformationThis project was funded by the US National Institutes of Health (NIA) and the Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proteolytic cleavage of vascular adhesion protein-1 induced by vascular endothelial growth factor in retinal capillary endothelial cells.

To investigate the mechanism of soluble vascular adhesion protein-1 (sVAP-1) accumulation induced by vascular endothelial growth factor (VEGF) in the vitreous of patients with diabetic retinopathy (DR). Experimental. Protein levels of sVAP-1 and N epsilon-(hexanoyl)lysine (HEL), an oxidative stress marker, in the vitreous samples from patients with proliferative diabetic retinopathy (PDR) with or without intravitreal bevacizumab (IVB) injection were determined by ELISA. The effect of VEGF on both mRNA expression of Vap-1 and secretion of sVAP-1 in rat retinal capillary endothelial cells (TR-iBRB2) was analyzed by real-time PCR and western blotting, respectively. In addition, the impact of VEGF on production and activation ratios of matrix metalloproteinase (MMP)-2 and MMP-9 was examined by gelatin zymography. Hydrogen peroxide production and reactive oxygen species (ROS) levels were assessed in the supernatants of TR-iBRB2 cells treated with VEGF. IVB injection decreased vitreous levels of sVAP-1 and HEL in patients with PDR. VEGF stimulation released sVAP-1 protein from TR-iBRB2 cells as a consequence of membrane-anchored VAP-1 shedding by MMP-2 and MMP-9. In addition, VEGF increased hydrogen peroxide generation and ROS augmentation through spermine oxidation by sVAP-1 as semicarbazide-sensitive amine oxidase (SSAO) in the supernatant of cultured endothelial cells. The current data demonstrate that proangiogenic factor VEGF induces sVAP-1 release from retinal capillary endothelial cells and facilitates hydrogen peroxide generation via enzymatic property of sVAP-1, followed by the increase of oxidative stress, one of the crucial factors in the pathogenesis of DR.

3D-Graphene supports for palladium nanoparticles: Effect of micro/macropores on oxygen electroreduction in Anion Exchange Membrane Fuel Cells

Hierarchically structured 3D-Graphene nanosheets as supports for palladium nanoparticles (Pd/3D-GNS) were fabricated using the Sacrificial Support Method. The pore size distribution of the 3D-GNS supports were tuned by utilizing smaller and larger sized sacrificial silica templates, EH5 and L90. Using a combination of Scanning Electron Microscopy (SEM), N2 sorption and Rotating Ring Disc Electrode (RRDE) technique, it was demonstrated that the EH5 and L90 modified 3D-GNS supports had higher percentage of micro- (<2 nm) and macropores (>50 nm), respectively. The templated pores also played a role in enhancing the oxygen reduction reaction (ORR) as well as membrane electrode assembly (MEA) performance of the Pd nanoparticles in comparison to non-porous 2D-GNS supports. Particularly, incorporation of micropores increased peroxide generation at higher potentials whereas presence of macropores increased both limiting current densities and reduce peroxide yields. Integration of the Pd/GNS nanocomposites into a H2/O2 fed Anion Exchange Membrane Fuel Cell (AEMFC) operating at 60°C also demonstrated the effect of modified porosity on concentration polarization or transport losses at high current densities. This strategy for the tunable synthesis of hierarchically 3D porous graphitized supports offers a platform for developing morphologically modified nanomaterials for energy conversion.

Denervated muscle fibers induce mitochondrial peroxide generation in neighboring innervated fibers: Role in muscle aging

Disruption of neuromuscular junctions and denervation of some muscle fibers occurs in ageing skeletal muscle and contribute to loss of muscle mass and function. Aging is associated with mitochondrial dysfunction and loss of redox homeostasis potentially occurs through increased mitochondrial generation of reactive oxygen species (ROS). No specific link between increased mitochondrial ROS generation and denervation has been defined in muscle ageing. To address this, we have examined the effect of experimental denervation of all fibers, or only a proportion of the fibers, in the mouse tibialis anterior (TA) muscle on muscle mitochondrial peroxide generation. Transection of the peroneal nerve of mice caused loss of pre-synaptic axons within 1–3 days with no significant morphological changes in post-synaptic structures up to 10 days post-surgery when decreased TA mass and fiber size were apparent. Mitochondria in the denervated muscle showed increased peroxide generation by 3 days post-transection. Use of electron transport chain (ETC) substrates and inhibitors of specific pathways indicated that the ETC was unlikely to contribute to increased ROS generation, but monoamine oxidase B, NADPH oxidase and phospholipase enzymes were implicated. Transection of one of the 3 branches of the peroneal nerve caused denervation of some TA muscle fibers while others retained innervation, but increased mitochondrial peroxide generation occurred in both denervated and innervated fibers. Thus the presence of recently denervated fibers leads to increased ROS generation by mitochondria in neighboring innervated fibers providing a novel explanation for the increased mitochondrial oxidative stress and damage seen with aging in skeletal muscles.

Acute ketamine impairs mitochondrial function and promotes superoxide dismutase activity in the rat brain.

Ketamine is often associated with altered mitochondrial function and oxidative stress. Nevertheless, limited data are still available regarding the in vivo action of ketamine in mitochondrial bioenergetics and redox state. Accumulating evidence supports a role for nitric oxide (NO) as a possible modulator of ketamine's side effects. In the present study, we investigated the role of NO modulation on ketamine anesthesia at the level of brain mitochondrial function and redox status. Adult male rats received a single dose of ketamine (50, 100, or 150 mg/kg IP) or a combination of ketamine and N-nitro-L-arginine (3 mg/kg IP). Animals were killed 6 hours after treatment. Brain and blood samples were collected for plasma NO determination and mitochondria isolation. Several variables of brain mitochondrial function were evaluated. Ketamine interfered with complex I function, revealing increased oxygen consumption in state 4, impaired oxidative phosphorylation efficiency of glutamate-malate substrate, and decreased NADH-ubiquinone oxidoreductase activity. In addition, mitochondrial NO synthase (mtNOS) activity and NO plasma levels were increased for the 50 and 100 mg/kg doses. Ketamine administration increased hydrogen peroxide generation and triggered superoxide dismutase activity. All these effects could totally or partially be prevented by mtNOS inhibition through N-nitro-L-arginine. Acute ketamine administration impaired the function of mitochondrial complex I leading to increased mtNOS activity, increased generation of hydrogen peroxide and NO, resulting in superoxide dismutase triggering, and improved antioxidant activity. The present findings clarify the role of NO modulation in ketamine anesthesia, providing new data on a relevant clinical mechanism.

음양곽(淫羊藿) 발효 추출물이 면역활성에 미치는 영향

ABSTRACT Objectives : This research aimed at studying the immuno modulating activity of Fermented Epimedii Herba (EHS).Method : The impacts on the cell viability, hydrogen peroxide and nitric oxide (NO) generation in cells, and cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1β, monocyte chemoattractant protein-1 (MCP-1) level have been measured by using Raw 264.7 cells with the specimen EHS as the fermented extract of Epimedii Herba with Saccharomyces cerevisiae STV89.Result : As a result of MTT assay to confirm the cytotoxicity of extrac ts from fermented Epimedii Herba, the toxicity was not excessively induced in Raw 264.7 cells when EHS were processed by concentration. EHS increased hydrogen peroxide generation in Raw 264.7 cells. EHS suppressed NO generation in Raw 264.7 cells while they significantly suppressed the increase of NO generati on induced by LPS in macrophage. EHS significantly decreased the generation amount of TNF-α and IL-6 induced by LPS in Raw 264.7 cells at 25 ㎍/mL or more.Conclusion : It appeared that the fermented extract of Epimedii Herba manuf actured from Epimedii Herba significantly has the immuno modulating acitivity as it did not excessively trigger cytotoxicity to Raw 264.7 cells, increased hydrogen peroxide generation in Raw 264.7 cell s, decreased NO generation in macrophage, and especially, suppressed both TNF-α and IL-6 generation in macrophage induced by LPS.Key words : Epimedii Herba, ferment, cytotoxicity, immuno modulating aciti vity, reactive oxygen species (ROS)

Hydrogen Peroxide Promotes Aging-Related Platelet Hyperactivation and Thrombosis

The incidence of thrombotic events increases during aging, but the mechanisms are not well understood. To investigate the prothrombotic role of oxidative stress during aging, we tested the hypothesis that aged mice overexpressing the antioxidant enzyme glutathione peroxidase-1 (Gpx1) are protected from experimental thrombosis. Susceptibility to carotid artery thrombosis was first examined in wild-type C57BL/6J mice. After photochemical injury of the carotid artery, the time to stable occlusion was significantly shorter in 12- and 18-month-old mice compared with 4-month-old mice (P<0.01). Unlike wild-type mice, transgenic mice overexpressing Gpx1 (Gpx1 Tg) did not exhibit shortened times to occlusion of the carotid artery at 12 or 18 months of age. Wild-type mice also exhibited increased susceptibility to venous thrombosis after inferior vena cava ligation at 12 or 18 months of age (P<0.05 versus 4 months of age). Gpx1 Tg mice were protected from this aging-related enhanced susceptibility to venous thrombosis. Age-dependent platelet hyperactivation, evidenced by increased hydrogen peroxide, fibrinogen binding, and activation of fibrinogen receptor αIIbβ3, was observed in thrombin-activated platelets from wild-type but not Gpx1 Tg mice (P<0.05). Enhanced platelet activation responses in aged mice were also prevented by polyethylene glycol-catalase or apocynin, an inhibitor of NADPH oxidase. Aged mice displayed increased intraplatelet expression of p47(phox) and superoxide dismutase-1, suggesting a mechanistic pathway for increased hydrogen peroxide generation. Our findings demonstrate that hydrogen peroxide is a key mediator of platelet hyperactivity and enhanced thrombotic susceptibility in aged mice.

Inorganic Polyphosphates Regulate Hexokinase Activity and Reactive Oxygen Species Generation in Mitochondria of Rhipicephalus (Boophilus) microplus Embryo

The physiological roles of polyphosphates (poly P) recently found in arthropod mitochondria remain obscure. Here, the possible involvement of poly P with reactive oxygen species generation in mitochondria of Rhipicephalus microplus embryos was investigated. Mitochondrial hexokinase and scavenger antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione reductase were assayed during embryogenesis of R. microplus. The influence of poly P3 and poly P15 were analyzed during the period of higher enzymatic activity during embryogenesis. Both poly Ps inhibited hexokinase activity by up to 90% and, interestingly, the mitochondrial membrane exopolyphosphatase activity was stimulated by the hexokinase reaction product, glucose-6-phosphate. Poly P increased hydrogen peroxide generation in mitochondria in a situation where mitochondrial hexokinase is also active. The superoxide dismutase, catalase and glutathione reductase activities were higher during embryo cellularization, at the end of embryogenesis and during embryo segmentation, respectively. All of the enzymes were stimulated by poly P3. However, superoxide dismutase was not affected by poly P15, catalase activity was stimulated only at high concentrations and glutathione reductase was the only enzyme that was stimulated in the same way by both poly Ps. Altogether, our results indicate that inorganic polyphosphate and mitochondrial membrane exopolyphosphatase regulation can be correlated with the generation of reactive oxygen species in the mitochondria of R. microplus embryos.

Mitochondrial-derived hydrogen peroxide inhibits relaxation of bovine coronary arterial smooth muscle to hypoxia through stimulation of ERK MAP kinase

Mitochondrial reactive oxygen species (ROS) are potentially important in vascular oxygen-sensing mechanisms because hypoxia appears to be a stimulus for mitochondrial ROS generation; however, scavenging of endogenous ROS does not alter relaxation of endothelium-denuded bovine coronary arteries (BCA) to hypoxia. The purpose of this study was to investigate the influence of increasing mitochondrial ROS on the relaxation of BCA to hypoxia. Increasing mitochondrial superoxide with inhibitors of electron transport (10 microM rotenone and antimycin) and by opening mitochondrial ATP-dependent K+ channels with 100 microM diazoxide were observed in this study to attenuate relaxation of BCA precontracted with 30 mM KCl to hypoxia by 68-76% and 38%, respectively. This effect of rotenone is not prevented by inhibiting NADPH oxidase (Nox) activation or scavenging superoxide with Peg-SOD; however, it is reversed 85% and 26% by increasing the consumption of intracellular peroxide by 0.1 mM ebselen and 32.5 U/ml Peg-catalase. Because inhibition of extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase (10 microM PD-98059), but not src kinase or rho kinase, also reverses the effects of rotenone by 69%, the peroxide-elicited force-enhancing effects of ERK appear to be attenuating the response to hypoxia. Rotenone increased the phosphorylation of ERK (by 163%). Activation of ERK in BCA with 0.1 mM peroxide or endogenous peroxide generated by stimulating Nox2 with a stretch treatment or contraction with 100 nM U-46619 also attenuated relaxation to hypoxia. Thus coronary arterial relaxation to hypoxia may be attenuated by pathophysiological conditions associated with increased peroxide generation by mitochondria or other sources that stimulate ERK.

Highlight Commentary on “Influence of lung oxidant and antioxidant status on alveolarization: Role of light-exposed total parenteral nutrition”

Bronchopulmonary dysplasia (BPD) is a frequent complication of premature newborns, particularly very low birth-weight babies (< 1500 g). Undoubtedly multiple mechanisms contribute to the adverse outcomes associated with BPD but oxidative stress is one causative factor. In this issue of Free Radical Biology and Medicine, Lavoie et al. describe the increased peroxide generation when the multivitamin solution used for nutritional support, total parenteral nutrition (TPN), is exposed to ambient light. Because the premature newborn has limited antioxidant capacity, this increased oxidative burden from the TPN becomes increasingly significant. Infusion of this light-exposed solution in a newborn guinea pig decreased lung tissue vitamin C but not vitamin E. When the multivitamin and lipid solutions were mixed and then exposed to light, alveolarization of the developing lung was decreased. This study by Lavoie et al. highlights simple measures that can potentially decrease the oxidant burden delivered to this vulnerable population and improve alveolarization.

Usnic acid-induced necrosis of cultured mouse hepatocytes: inhibition of mitochondrial function and oxidative stress

Usnic acid, a lichen acid, is a compound found in crude medicines and dietary supplements, including Lipokinetix ®, a supplement marketed as a weight loss agent that caused hepatotoxicity and acute liver failure in patients. In this study, we examined the toxicity of usnic acid and assessed whether usnic acid may be contributing to hepatotoxicity caused by Lipokinetix. In primary cultured murine hepatocytes, usnic acid treatment (5 μM) resulted in 98% necrosis within 16 hr (no apoptosis was detected). Usnic acid treatment was associated with early inhibition and uncoupling of the electron transport chain in mitochondria of cultured hepatocytes. This inhibition of mitochondria by usnic acid corresponded with a fall in ATP levels in hepatocytes. In isolated liver mitochondria, usnic acid was observed to directly inhibit and uncouple oxidative phosphorylation. Oxidative stress appears to be central in usnic acid-induced hepatotoxicity based on the following findings: (1) pretreatment with antioxidants (butylated hydroxytoluene + Vitamin E) decreased usnic acid-induced necrosis by nearly 70%; (2) depletion of mitochondrial GSH with diethylmaleate increased susceptibility of hepatocytes to usnic acid; (3) usnic acid treatment was associated with increase free radical generation, measured using the fluorescent probe, dichlorodihydrofluorescin. The source of reactive oxygen species after usnic acid treatment include autoxidation of usnic acid and increased hydrogen peroxide generation by mitochondria caused by usnic acid inhibition of the respiratory chain, with the latter playing a more prominent role. Taken together, our results suggest that usnic acid is a strong hepatotoxic agent that triggers oxidative stress and disrupts the normal metabolic processes of cells. Usnic acid therefore may contribute to the hepatotoxic effects of Lipokinetix and its use in any supplement must come into question.

Reversibility of leukocyte dysfunction in rats with obstructive jaundice

Background The role of leukocytes in obstructive jaundice is obscure and the effect of relieving cholestasis on leukocyte function is unclear. We postulated that cholestasis affects systemic polymorphonuclear leukocyte function by deranging phagocytosis and hydrogen peroxide release and the leukocyte dysfunction is reversible by internal and external biliary drainage. Materials and methods Sixty male Sprague Dawley rats were randomly assigned to four groups: obstructive jaundice (OJ), sham operation (SH), OJ with internal drainage (ID), and OJ with external drainage (ED). The phagocytic functions of neutrophils and monocytes in whole blood were measured with flow cytometry using fluorescent microspheres. Intracellular hydrogen peroxide production by leukocytes was assessed with flow cytometry using dihydrorhodamine-123 as probes. Results Leukocyte count and percentage of monocytes in rats with OJ was significantly increased compared with SH rats ( P < 0.001). These elevations could be reversed by both ID and ED method ( P < 0.001). The phagocytic function of neutrophils and monocytes was significantly depressed in OJ rats compared with that in SH rats ( P < 0.001). After relief of the OJ, the suppressed phagocytic function of neutrophils and monocytes was completely improved in ID rats (ID versus OJ, P < 0.001), but only partially reversed in ED rats. The hydrogen peroxide production by monocytes and lymphocytes was significantly increased in OJ rats ( P < 0.05). ID reversed the increased hydrogen peroxide generation ( P < 0.05), but ED only partially did. Conclusions In our rodent model of biliary obstruction, deranged phagocytosis, and hydrogen peroxide generation by leukocytes was found. Internal drainage is superior to external drainage for reversal of the distorted leukocyte function.

Increased hydrogen peroxide generation by neutrophils from patients with acne inflammation.

Reactive oxygen species generated by neutrophils are closely correlated with the pathogenesis of a variety of inflammatory skin diseases. The aim of this study was to investigate the possible role of reactive oxygen species generated by neutrophils in the mediation of acne inflammation. Bacterial phagocytotic stimuli, mediated by opsonin activity, were applied to whole blood, and neutrophil hydrogen peroxide production was measured. Patients with acne inflammation showed a significantly increased level of hydrogen peroxide produced by neutrophils compared to patients with acne comedones and healthy controls. There were no marked differences in the level of hydrogen peroxide produced by neutrophils between patients with acne comedones and healthy controls. In addition, patients with acne inflammation treated by oral administration of minocycline hydrochloride, a drug that inhibits hydrogen peroxide generation by neutrophils, showed a significant decrease in the ability of neutrophils to produce hydrogen peroxide in accordance with a decrease in the inflammatory activity of acne lesions. The present study seems to suggest that acne inflammation is mediated in part by hydrogen peroxide generation by neutrophils.

Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae.

We have analyzed the proteins that are oxidatively damaged when Saccharomyces cerevisiae cells are exposed to stressing conditions. Carbonyl groups generated by hydrogen peroxide or menadione on proteins of aerobically respiring cells were detected by Western blotting, purified, and identified. Mitochondrial proteins such as E2 subunits of both pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, aconitase, heat-shock protein 60, and the cytosolic fatty acid synthase (alpha subunit) and glyceraldehyde-3-phosphate dehydrogenase were the major targets. In addition we also report the in vivo modification of lipoamide present in the above-mentioned E2 subunits under the stressing conditions tested and that this also occurs with the homologous enzymes present in Escherichia coli cells that were used for comparative analysis. Under fermentative conditions, the main protein targets in S. cerevisiae cells treated with hydrogen peroxide or menadione were pyruvate decarboxylase, enolase, fatty acid synthase, and glyceraldehyde-3-phosphate dehydrogenase. Under the stress conditions tested, fermenting cells exhibit a lower viability than aerobically respiring cells and, consistently, increased peroxide generation as well as higher content of protein carbonyls and lipid peroxides. Our results strongly suggest that the oxidative stress in prokaryotic and eukaryotic cells shares common features.

DNA damage related to increased hydrogen peroxide generation by hypolipidemic drug-induced liver peroxisomes.

Several hypolipidemic drugs and certain industrial plasticizers induce proliferation of peroxisomes, enhance the activity of peroxisome-associated beta-oxidation of fatty acids, and produce hepatocellular carcinomas in the livers of rodents. Because these chemicals themselves are not mutagens and do not covalently modify DNA, unlike the majority of chemical carcinogens, we proposed that the persistent proliferation of peroxisomes, and the induction of associated peroxisomal oxidases, caused a sustained increase in intracellular H2O2 or other reduced oxygen species, which would then introduce mutagenic DNA damage. In the present study, we investigated the ability of peroxisomes purified from the livers of normal and hypolipidemic drug-treated rats to induce DNA strand scission in vitro. Gradient-purified peroxisomes from livers of hypolipidemic drug-treated rats produced a 30- to 70-fold increase in H2O2 generation when compared to controls. The levels of H2O2 generated in incubations containing control or hypolipidemic drug-induced peroxisomes correlated well with the induction of single strand breaks in supercoiled simian virus 40 DNA molecules that were included in these reconstituted peroxisome incubations. Addition of excess catalase to peroxisome incubations failed to prevent strand breaks, suggesting that other reduced oxygen species may be rapidly generated from H2O2. These experimental results are consistent with a mechanism of hepatocarcinogenesis in which hepatocellular genetic damage is introduced by the by-products of peroxisomal fatty acid beta-oxidation, an oxidative pathway that is dramatically increased in hypolipidemic drug-treated livers.