Peroxide Toxicity Research Articles

Neuritogenic and Neuroprotective Activities of Fruit Residues

Neuritogenic and neuroprotective activities of litchi (Litchi chinensis Sonn., Sapindaceae) and salacca (Salacca edulis Reinw., Arecaceae) pericarp, and sapodilla (Achras sapota L., Sapotaceae) and tamarind Srichompu cultivar (Tamarindus indica L., Caesalpiniaceae) seed coat extracts were evaluated on cultured cholinergic P19-derived neurons. All the extracts, at a very low concentration (1 ng/mL of litchi and salacca pericarp extracts, 10 ng/mL of sapodilla and 100 ng/mL of tamarind seed coat extracts), enhanced the survival of cultured neurons (% viability more than 100%) by XTT reduction assay. The extracts were further evaluated for their neuritogenicity by observing cell morphology by phase-contrast microscopy and neuroprotective activity in serum deprivation and pre- and co-administration of hydrogen peroxide models. The phase-contrast micrographs displayed that all of the extracts possessed neurogenic activity by promoting the neurite outgrowth of the cultured neurons. Moreover, these extracts can protect neurons from oxidative stress-caused cell death in a serum deprivation model, and prevent and protect neuron cells from the toxicity of hydrogen peroxide. In this study we assured that the neuritogenic and neuroprotective activities of these extracts derived from the phenolic components and flavonoids contained in the extracts by acting as signaling molecules to enhance neuron survival and promote neurite outgrowth. These results suggest that all of the extracts are potentially sources of neuritogenic and neuroprotective components which might be used either as pharmaceutical products or dietary supplements for neurodegenerative disorder patients, for example, those suffering from Alzheimer's disease.

Methionine sulphoxide reductases revisited: free methionine as a primary target of H2O2 stress in auxotrophic fission yeast

Amino acid methionine can suffer reversible oxidation to sulphoxide and further irreversible over-oxidation to methionine sulphone. As part of the cellular antioxidant scavenging activities are the methionine sulphoxide reductases (Msrs), with a reported role in methionine sulphoxide reduction, both free and in proteins. Three families of Msrs have been described, but the fission yeast genome only includes one representative for two of these families: MsrA/Mxr1 and MsrB/Mxr2. We have investigated their role in methionine reduction and H2 O2 sensitivity. We show here that MsrA/Mxr1 is able to reduce free oxidized methionine. Cells lacking each one of the genes are not significantly sensitive to different types of oxidative stresses, neither display altered life span. However, only when deletion of msrA/mxr1 is combined with deletion of met6, which confers methionine auxotrophy, the survival upon H2 O2 stress decreases by 100-fold. In fact, cells lacking only Met6, and which therefore require addition of methionine to the growth media, are extremely sensitive to H2 O2 stress. These and other evidences suggest that oxidation of free methionine is a primary target of peroxide toxicity in cells devoid of methionine biosynthetic capacity, and that an important role of Msrs is to recycle this oxidized free amino acid.

Cat’s whiskers flavonoid attenuated oxidative DNA damage and acute inflammation: its importance in lymphocytes of patients with rheumatoid arthritis

Cleome gynandra L. (Capparidaceae) is one of the vegetables commonly known as 'Hurhur' and 'Karaila' in India, 'Pe Hua Tsai' in China and "Cat's whiskers" in English. Present study was aimed to characterize previously isolated Cat's whiskers flavonoid as 5-hydroxy-3, 7, 4' -trimethoxyflavone (5HTMF) and to evaluate its effect on carrageenan-induced acute inflammation in rats and hydrogen peroxide induced DNA damage in mouse macrophages. The ex vivo effect of 5HTMF upon generation of free radicals in the mononuclear lymphocytes of patients with rheumatoid arthritis (RA) was also evaluated. 5HTMF not only reduce the swelling of hind paw in rats from 1 to 3h of carrageenan injection but also decreased serum nitric oxide (NO) production. Toxic hydrogen peroxide induced oxidative DNA damage that was significantly decreased by 5HTMF. Though oxidative stress is a potential biomarker for determining disease activity in patients with RA, surprisingly 5HTMF inhibited the superoxide, hydroxyl and NO radicals in the isolated peripheral blood mononuclear lymphocytes of patients with RA. From the above study, it may be concluded 5HTMF attenuated acute inflammation by inhibiting NO and by protecting the oxidative DNA damage due to hydrogen peroxide scavenging property. It was also equally effective in scavenging the free radicals in lymphocytes of patients with RA. Collectively, our results indicate that 5HTMF as well as leafy vegetable of Cat's whiskers may be a promising nontoxic food alternative in attenuating the oxidative stress, meriting further studies on other human inflammatory cells.

Short communication: Acute toxicity of hydrogen peroxide in juvenile white shrimp Litopenaeus vannamei reared in biofloc technology systems

Biofloc technology (BFT) has been used to rear white shrimp, Litopenaeus vannamei. In this culturing system, the absence of aeration causes a rapid drop in dissolved oxygen levels, and hydrogen peroxide (H2O2) can be used as an emergency source of oxygen. This study aimed to determine the lethal concentration and safe level of H2O2 applied as a source of oxygen for juvenile white shrimp L. vannamei in a BFT system. Juveniles (1.39 ± 0.37 g) were exposed for 2 h to different concentrations of H2O2 [29 (100), 58 (200), 116 (400), 174 (600), 232 (800), 290 (1,000) and 348 (1,200) μL H2O2/L (ppm H2O2-29 %/L)] in addition to a control group without addition of H2O2, and the survival rates were monitored for 96 h. The LC50 values and 95 % confidence intervals at 24, 48, 72 and 96 h were 235.5 (207–268), 199.1 (172–229), 171.1 (146–198) and 143.3 (120–170) μL H2O2/L, respectively. The safe level was 14.3 μL H2O2/L, and the highest concentration with survival rates similar to the control group (NOAEC) was 29 μL H2O2/L. In these concentrations, H2O2 can be used as a safe source of oxygen for L. vannamei reared in BFT systems.

English

Introduction Chronic inflammation is a prolonged pathological condition characterised by mononuclear cell infiltration, tissue destruction and fibrosis due to excess production of free radicals. Rheumatoid arthritis is systemic autoimmune disease characterised by synovial cell infiltration as well as destruction of tissues. Free radicals are defined as molecules or any chemical species having an unpaired electron in the outer orbit and are capable of independent existence. In biological systems, the most common source of free radicals is oxygen which in term reacts with nitrogen and produces nitrogen intermediates. Under certain stress conditions, Oxygen becomes much more active and forms superoxide anion radicals and hydroxyl radical (OH−). The important nitrogen species are nitric oxide and peroxynitrite anion. Nicotinamide adenine dinucleotide phosphate oxidation by Nicotinamide adenine dinucleotide phosphate oxidase is considered as the major source of Superoxide anion radicals, where the antioxidant enzyme superoxide dismutase converts Superoxide anion radicals to less toxic hydrogen peroxide. Nitric oxides are formed from L-arginine by one of the three nitric oxide synthases Free radical biology in cellular inflammation related to rheumatoid arthritis A Bala1,2*, PK Haldar1 and react with O2 and produce highly toxic Peroxynitrite. Collectively all reactive oxygen and nitrogen species are ultimately responsible for creating oxidative stress during inflammation by producing different cytotoxic cytokines through the phosphorylation of nuclear factor kappa-B (NFκB). The main aim of this review is to analyse the role of free radicals in cellular inflammation, related to rheumatoid arthritis. Conclusion DNA damage caused by reactive radicals as well as cytokines may lead to abnormal functioning of the cells. Although cellular repair systems correct most of these damages but the free radicals induced DNA lesions can be an important aetiology of inflammatory autoimmune diseases like rheumatoid arthritis. Thus it should be recommended that management of oxidative stress by antioxidant therapies in combination with modulators of cytokines and other inflammatory pathways are the therapeutic weapon to fight against rheumatoid arthritis. Introduction Inflammation is a protective defence mechanism employed by cells/ tissues against endogenous and exogenous stimuli/antigens1. The relationship between chronic inflammation and rheumatoid arthritis (RA) has already been recognised2. RA is a systemic autoimmune disorder that primarily targets the synovium of diarthrodial joints resulting in an unchecked synovial inflammation3,4 and accumulation of inflammatory cells like monocytes, macrophages and neutrophils in sinovium. In acute inflammatory processes there is a marked accumulation of polymorphonuclear neutrophills however, chronic inflammation is a prolonged pathological condition characterised by mononuclear immune cell infiltration, tissue destruction and fibrosis due to excess production of reactive oxygen and nitrogen species (ROS and RNS) as well as toxic free radicals5. Chronic inflammation exerts its cellular side effects mainly through excessive production of free radicals and depletion of antioxidant defence in the body6. The main aim of this review is to analyse the role of free radicals in cellular inflammation, related to RA. What is free radical? In the general structure of atoms and molecules, electrons are usually associated and exist in pairs, each pair moving within a defined region of space (an atomic or molecular orbital). One electron in each pair has a spin quantum number of + 1⁄2, the other − 1⁄27. Free radicals are defined as molecules or any chemical species with an unpaired electron in the outer orbit and are capable of independent existence (hence the term ‘free’)8. This unpaired electron usually produces a highly reactive free radical. In biological systems, the most common source of free radicals is oxygen which in term reacts with nitrogen and produces nitrogen intermediates and is termed as ROS and RNS9. Under certain stress conditions, Oxygen becomes much more active and forms ROS, such as superoxide anion radicals (O2), hydroxyl radical (OH−), peroxyl radical (ROO−) * Corresponding author Email: asisbala_ju@yahoo.co.in 1 Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, West Bengal, India 2 Guru Nanak Institute of Pharmaceutical Science and Technology, 157/F Nillgunj Road, Panihati, Sodepur, Kolkata 700114, West Bengal, India

Elucidating Alzheimer’s disease neuronal death by the ‘calpain-cathepsin hypothesis'

Alzheimer's disease (AD) is characterized by slowly progressive neuronal death, but its molecular cascade remains elusive for over 100 years. Since accumulation of autophagic vacuoles (granulo-vacuolar degenerations) represents one of the histological hallmarks of degenerating neurons in AD, a causal link between autophagy failure and neuronal death is probable. Recent data advocate for dual roles of heat-shock protein 70.1 (Hsp70.1) as a molecular chaperone for damaged proteins and a guardian of lysosomal integrity. The aim of this study is at elucidating the underlying mechanism of AD by comparing ischemic neuronal death of monkeys and degenerative neuronal death of AD patients. Morphologic, immunoblotting, and proteomics analyses of both the ischemic monkey (Macaca fuscata, body weight of 5–10 Kg, n=20) and human AD brains were done. The ischemic monkey data in the author's laboratory were compared to the human AD patient data of the literature. The main topics are whether an age-related decrease in lysosomal and autophagic activities has a causal connection to programmed neuronal necrosis in sporadic AD, and how genetic factors such as apolipoprotein E (APOE) and presenilin 1 can facilitate lysosomal de stabilization. Both monkey and human brains exhibited evidence of transient or continual perturbed Ca 2+ homeostasis, calpain activation, and oxidative stresses upon Hsp70.1. Membrane lipids such as linoleic and arachidonic acids are vulnerable to the cumulative oxidative stresses, generating a toxic peroxidation product 'hydroxynonenal' that carbonylated Hsp70.1. In the monkey brain, both the carbonylation and calpain-mediated cleavage occurred at Arg469 (i.e., key site) of Hsp70.1. This caused decrease of acid sphingomyelinase activity and accumulation of lysosomal sphingomyelin. Electron microscopic data of the AD neurons showed evidence of lysosomal destabilization and multilamellar lysosomal lipidosis. Taken together, impairments of lysosomal autophagy and stabilization should be driven by the calpain-mediated cleavage of carbonylated Hsp70.1, and this causes lysosomal permeabilization and/or rupture with the resultant release of the cell degradation enzyme, cathepsins (calpain-cathepsin hypothesis: Yamashima T., J Neurochem. 120:477–494, 2012).

Pepino mosaic virus triple gene block protein 1 (TGBp1) interacts with and increases tomato catalase 1 activity to enhance virus accumulation

Various plant factors are co-opted by virus elements (RNA, proteins) and have been shown to act in pathways affecting virus accumulation and plant defence. Here, an interaction between Pepino mosaic virus (PepMV) triple gene block protein 1 (TGBp1; p26) and tomato catalase 1 (CAT1), a crucial enzyme in the decomposition of toxic hydrogen peroxide (H₂O₂), was identified using the yeast two-hybrid assay, and confirmed via an in vitro pull-down assay and bimolecular fluorescent complementation (BiFC) in planta. Each protein was independently localized within loci in the cytoplasm and nuclei, sites at which their interaction had been visualized by BiFC. Following PepMV inoculation, CAT mRNA and protein levels in leaves were unaltered at 0, 3 and 6 days (locally) and 8 days (systemically) post-inoculation; however, leaf extracts from the last two time points contained increased CAT activity and lower H₂O₂ evels. Overexpression of PepMV p26 in vitro and in planta conferred the same effect, suggesting an additional involvement of TGBp1 in potexvirus pathogenesis. The accumulation of PepMV genomic and subgenomic RNAs and the expression of viral coat protein in noninoculated (systemic) leaves were reduced significantly in CAT-silenced plants. It is postulated that, during PepMV infection, a p26-CAT1 interaction increases H₂O₂ cavenging, thus acting as a negative regulator of plant defence mechanisms to promote PepMV infections.

Development of a fluorescence analysis method for N-acetylneuraminic acid and its oxidized product ADOA

N-acetylneuraminic acid (NANA) consumes toxic hydrogen peroxide (H2O2) under physiological conditions and is oxidized by an equimolar amount of H2O2 to yield its decarboxylated product 4-(acetylamino)-2,4-dideoxy-d-glycero-d-galacto-octonic acid (ADOA). Highly sensitive analytical methods are required to detect ADOA in the human body. We labeled NANA and ADOA with 4-(N,N-dimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DBD-ED) to enable their fluorometric detection, and developed a method using HPLC with fluorometric detection (HPLC-FD) for the simultaneous determination of the derivatized NANA and ADOA. The derivatized NANA and ADOA were separated by a hydrophilic interaction liquid chromatography (HILIC) column using an H2O/CH3CN/HCOOH (10/90/0.35) mobile phase. Fluorescence was monitored at excitation and emission wavelengths of 450nm and 560nm, respectively. Both intra- and inter-day (n=6) repeat determinations of the DBD-ED-derivatized NANA and ADOA gave relative standard deviations of less than 5%. The calibration curves for standard solutions of DBD-ED-derivatized NANA and ADOA were linear over the ranges from 576fmol to 2.0nmol and 556fmol to 2.0nmol, respectively. The method developed was highly specific and sensitive for NANA and ADOA. The presence of ADOA in biological samples was revealed for the first time using this method.

Myeloperoxidase Deficiency: The Secret Under the Flag of Unstained Cell

Myeloperoxidase (MPO) deficiency is one of the most common inherited phagocyte defects, but it is rarely associated with clinical symptoms [1]. MPO, which is abundant in azurophilic granules of neutrophils and in the lysosomes of monocytes, plays a key role in amplifying the toxicity of hydrogen peroxide generated by the respiratory burst [1,2]. The diagnosis of MPO deficiency was rare before 1979; currently, the diagnosis is easily made due to the widespread use of automated flow cytochemical analysis in clinical hematology laboratories for enumerating peripheral blood neutrophils with peroxidase activity [2]. The MPO gene is encoded on chromosome 17q23 [3]. Primary deficiency of MPO is inherited as an autosomal recessive disorder. A secondary form of MPO deficiency has been described in various disorders, including lead poisoning, severe infections, neuronal lipofuscinosis, obstructive jaundice, diabetes mellitus, and such disseminated cancers as acute and chronic myeloid leukemia, myelodysplastic syndrome, and Hodgkin’s lymphoma [1,2,4]. MPO-deficient neutrophils are markedly less efficient at killing Candida albicans and Aspergillus hyphae [1]. Although MPO is involved in killing certain microorganisms, to date, no particular susceptibility to persistent or severe infections has been noted in the vast majority of MPO-deficient patients [1,2]. Recurrent Candida infection has been observed predominantly in MPO-deficient patients that also have diabetes mellitus. In addition to infectious manifestations, the relationship between congenital MPO deficiency and the occurrence of malignancy remains controversial [1,2]. There is no specific treatment for MPO deficiency; however, in symptomatic patients antifungal treatment is crucial. Herein we present a 17-year-old male that presented to our hematology department with neutropenia. Anamnesis showed that the patient did not have any recurrent or severe infection, but had been receiving benzathine penicillin prophylaxis due to acute rheumatic fever for years. Physical examination showed the following: weight: 58 kg (3-10p); height: 169 cm (10-25p); 1-2-degree/6 systolic murmur in the mesocardial region; the remainder of the examination was unremarkable. Complete blood count results were as follows: hemoglobin: 13.1 g/dL; leucocyte count: 6140/mm3; platelet count: 503,000/mm3. The formulation of the leucocytes was interesting, with an absolute neutrophil count of 20/mm3 and a high absolute unstained cell count of 3020/mm3 (nearly 50%). Although the absolute neutrophil count was 20/mm3, peripheral blood smear showed normal formulation of leucocytes (49% neutrophils, 47% lymphocytes, and 4% monocytes). The inconsistency between the absolute neutrophil count and the peripheral blood smear suggested the possibility of MPO deficiency, and we therefore performed cytochemical staining of peroxidase and flow cytometry analysis. Cytochemical staining of peroxidase was negative, as compared to the control (Figure 1a, 1b), and flow cytometry analysis showed a dramatic decrease in MPO(+) neutrophils (Figure 2). In addition, we identified a compound heterozygous MPO gene mutation [c.1705C> T];[c.2031-2A>C]. The patient was followed-up for 12 months after his initial examination, during which time he was asymptomatic and healthy. In conclusion, we think that MPO deficiency must be considered in patients with incompatibility of neutropenia between smear and counter, especially when the flag of unstained cell is remarked. Figure 1 The cytochemical staining of the neutrophil from patient (a) shows negativity when compared to the control (b). Figure 2 The flow cytometry analysis showed reduced positivity of myeloperoxidase at the gate of neutrophils.

Lipophilic adamantyl- or deferasirox-based conjugates of desferrioxamine B have enhanced neuroprotective capacity: implications for Parkinson disease

Parkinson disease (PD) is a neurodegenerative disease characterized by death of dopaminergic neurons in the substantia nigra region of the brain. Iron content is also elevated in this region in PD and is implicated in the pathobiology of the disease. Desferrioxamine B (DFOB) is a high-affinity iron chelator and has shown efficacy in animal models of Parkinson disease. The high water solubility of DFOB, however, attenuates its ability to enter the brain. In this study, we have conjugated DFOB to derivatives of adamantane or the clinical iron chelator deferasirox to produce lipophilic compounds designed to increase the bioavailability of DFOB to brain cells. We found that the novel compounds are highly effective in preventing iron-mediated paraquat and hydrogen peroxide toxicity in neuronal-like BE2-M17 dopaminergic cells, primary neurons, and iron-loaded or glutathione-depleted primary astrocytes. The compounds also alleviated paraquat toxicity in BE2-M17 cells that express the PD-causing A30P mutation of α-synuclein. This protection was ∼66-fold more potent than DFOB alone and also more effective than other cell-permeative metal chelators, clioquinol and phenanthroline. These results demonstrate that increasing the bioavailability of DFOB through the conjugation of lipophilic fragments greatly enhances its protective capacity. These novel compounds have potential as therapeutics for the treatment of PD and other conditions of Fe dyshomeostasis.

Increased vulnerability of parkin knock down PC12 cells to hydrogen peroxide toxicity: The role of salsolinol and NM-salsolinol

Dopamine-derived neurotoxins, 1-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) and 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (NM-salsolinol) are the two most possible 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-like endogenous neurotoxin candidates that involved in the pathogenesis of Parkinson’s disease (PD). The levels of endogenously synthesized salsolinol and NM-salsolinol are increased in the cerebrospinal fluid (CSF) of PD patients. Both of them lead to neurotoxicity in dopaminergic cells by inhibiting mitochondrial electron transport chain. To study the role of salsolinol and NM-salsolinol in Parkin deficiency-induced dopaminergic cell damage, we determined the cellular level of oxidative stress, the formation of salsolinol and NM-salsolinol, the level of mitochondrial damage and cell viability with/without the presence of exogenous H2O2 using differentiated dopaminergic PC12 cells. Our data show that parkin knock down elevates cellular oxidative stress, salsolinol and NM-salsolinol levels, which are responsible for the higher cell mortality in Parkin-deficient cells upon exposure to exogenous H2O2. The level of mitochondrial membrane potential loss, cristae disruption and the release of cytochrome c increased significantly along with the increased level of salsolinol and NM-salsolinol, whereas compared to parkin knock down cells in the presence of H2O2, the mitochondrial damage and higher cell mortality were both diminished when the levels of salsolinol and NM-salsolinol was reduced. The results not only indicate the elevated level of salsolinol and NM-salsolinol, but also reveal the potential role of salsolinol and NM-salsolinol in parkin knock down-induced cell vulnerability. We assume that parkin deficiency is the trigger of excessive oxidative stress, elevated endogenous neurotoxin levels and mitochondrial damage, which eventually results in cell death of dopaminergic cells.

Enhanced 4-Hydroxynonenal Resistance inKEAP1Silenced Human Colon Cancer Cells

Nuclear factor erythroid 2-related factor 2 (NRF2) is the transcription factor that regulates an array of antioxidant/detoxifying genes for cellular defense. The conformational changes of Kelch-like ECH-associated protein 1 (KEAP1), a cytosolic repressor protein of NRF2, by various stimuli result in NRF2 liberation and accumulation in the nucleus. In the present study, we aimed to investigate the effect of KEAP1 knockdown on NRF2 target gene expression and its toxicological implication using human colon cancer cells. The stable KEAP1-knockdown HT29 cells exhibit elevated levels of NRF2 and its target gene expressions. In particular, the mRNA levels of aldo-keto reductases (AKR1C1, 1C2, 1C3, 1B1, and 1B10) were substantially increased in KEAP1 silenced HT29 cells. These differential AKRs expressions appear to contribute to protection against oxidative stress. The KEAP1-knockdown cells were relatively more resistant to hydrogen peroxide (H2O2) and 4-hydroxynonenal (4HNE) compared to the control cells. Accordantly, we observed accumulation of 4HNE protein adducts in H2O2- or 4HNE-treated control cells, whereas KEAP1-knockdown cells did not increase adduct formation. The treatment of KEAP1-silenced cells with AKR1C inhibitor flufenamic acid increased 4HNE-induced cellular toxicity and protein adduct formation. Taken together, these results indicate that AKRs, which are NRF2-dependent highly inducible gene clusters, play a role in NRF2-mediated cytoprotection against lipid peroxide toxicity.

НАРУШЕНИЯ ЭНЕРГЕТИЧЕСКОГО МЕТАБОЛИЗМА ВО ВНУТРЕННИХ ОРГАНАХ ПРИ ЧЕРЕПНО-МОЗГОВОЙ ТРАВМЕ НА ФОНЕ САХАРНОГО ДИАБЕТА

Traumatic cranial injury is one of the leading reasons in the statistics of mortality. Modern ideas about the pathogenesis of traumatic brain injury based on the allocation of primary and secondary factors of brain damage. One of the main causes of secondary brain injury is hypoxia – insufficient provision with oxygen. Changes in oxygen balance lead to activation of oxidative stress, disturbances of mitochondria membranes and their death, and progression of energy metabolism imbalance caused by trauma. Millions of people worldwide suffer from diabetes mellitus. One of the factors that influences the development of complications of diabetes, is an accumulation of highly toxic peroxide compounds that intensify the processes of destabilization of cell membranes. Further research of peculiarities of oxidative stress and energy metabolism imbalance in diabetes associated with trauma will provide the basis for therapeutic directions to increase treatment options and improve patient health and well-being.Therefore, the purpose of this work is to study the effect of traumatic cranial injury on the energy supplying processes in the liver and kidneys of rats with streptozotocin diabetes, as well as establishing a link between impaired mitochondrial oxidation and the intensity of oxidative stress.Hundred adult male Wistar rats, weighing 180-220 g, were used in this study. Rats were assigned to one of four groups: (1) control animals (n=10); (2) rats with diabetes (n=10); (3) rats with closed cranial trauma (n=40); (4) animals with cranial trauma on the diabetes background (n=40). To induce diabetes, streptozotocin (“Sigma-Aldrich”,USA) was dissolved in citrate buffer (pH 4.5) and a single intra-peritoneal injection (60 mg/kg) was given to each animal. The application of the cranial injury was done according to the method developed in our laboratory. The rats were sacrificed under thiopental sodium anesthesia by total bloodletting from the heart. The rats of groups 3 and 4 were euthanized 3 h, 24 h, 5, and 14 days after the onset of the injury. Blood of the experimental animals was analyzed for succinate dehydrogenase, cytochrome oxidase, proton adenosine triphosphatase activity, the concentration of adenosine triphosphate, diphosphate, and monophosphate. Additionally, we studied the products of protein and lipid peroxidation.Succinate dehydrogenase activity decreased 22,6% after 3 h, by 38,8% after 24 h, by 34,7% after 5 days, and by 22,2% after 14 days post-traumatically in diabetic rats. The most pronounced decrease in cytochrome oxidase activity in the liver was 26,3 and 40 % after 5 and 14 days after TBI, respectively. These changes were accompanied by an increase in the proton adenosine triphosphatase activity in the liver by 42,1, 53,5, 68,6, 63,3 % after 3 h, 1, 5 and 14 days after injury, compared to injured rats without diabetes. Statistically significant increase in the products of protein and lipid peroxidation after traumatic cranial injury in combination with diabetes mellitus towards non-diabetic injured rats was observed.It has been established that there are inhibition of the respiratory chain enzymes activity (succinate dehydrogenase and cytochrome oxidase), an increase in the proton adenosine triphosphatase activity and a decrease in the concentration of adenosine triphosphate in the liver and kidneys of experimental animals with cranial trauma and diabetes. The degree of mitochondrial oxidation impairment and adenosine triphosphate synthesis processes positively correlates with the intensity of oxidative stress in the liver and kidneys. In the event of traumatic cranial injury on the background of concomitant diabetes mellitus, indicators of the mitochondrial energy supplying oxidation, the level of macroergic compounds and the intensity of oxidative stress were significantly worse compared to analogical indicators in the normoglycemic injured animals.

Resveratrol attenuates L-DOPA-induced hydrogen peroxide toxicity in neuronal cells

A variety of polyphenol antioxidant compounds derived from natural products have demonstrated neuroprotective activity against neuronal cell death. The objective of this study was to investigate the effect of resveratrol (RESV) and bioflavonoids in attenuating hydrogen peroxide (H(2)O(2))-induced oxidative stress in neuronal cells. H2O2 levels were increased by the addition of L-3,4-dihydroxyphenylalanine (L-DOPA) to cultured dopaminergic SKNSH cells. H(2)O(2) was monitored by peroxyfluor-1, a selective H(2)O(2) optical probe. To examine the neuroprotective effects of RESV and bioflavonoids against L-DOPA, we cotreated RESV, quercetin, or (-) epigallocatechin gallate with L-DOPA and monitored for H(2)O(2) levels. The combination of RESV and L-DOPA was 50% more effective at reducing H(2)O(2) levels than the combination of quercetin or epigallocatechin gallate with L-DOPA. However, the combination of each antioxidant with L-DOPA was effective at preserving cell viability.

Distinct Characteristics of Two 2-Cys Peroxiredoxins of Vibrio vulnificus Suggesting Differential Roles in Detoxifying Oxidative Stress

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes reducing toxic peroxides. Two distinct 2-Cys Prxs, Prx1 and Prx2, were identified in Vibrio vulnificus, a facultative aerobic pathogen. Both Prxs have two conserved catalytic cysteines, C(P) and C(R), but Prx2 is more homologous in amino acid sequences to eukaryotic Prx than to Prx1. Prx2 utilized thioredoxin A as a reductant, whereas Prx1 required AhpF. Prx2 contained GGIG and FL motifs similar to the motifs conserved in sensitive Prxs and exhibited sensitivity to overoxidation. MS analysis and C(P)-SO(3)H specific immunoblotting demonstrated overoxidation of C(P) to C(P)-SO(2)H (or C(P)-SO(3)H) in vitro and in vivo, respectively. In contrast, Prx1 was robust and C(P) was not overoxidized. Discrete expression of the Prxs implied that Prx2 is induced by trace amounts of H(2)O(2) and thereby residential in cells grown aerobically. In contrast, Prx1 was occasionally expressed only in cells exposed to high levels of H(2)O(2). A mutagenesis study indicated that lack of Prx2 accumulated sufficient H(2)O(2) to induce Prx1. Kinetic properties indicated that Prx2 effectively scavenges low levels of peroxides because of its high affinity to H(2)O(2), whereas Prx1 quickly degrades higher levels of peroxides because of its high turnover rate and more efficient reactivation. This study revealed that the two Prxs are differentially optimized for detoxifying distinct ranges of H(2)O(2), and proposed that Prx2 is a residential scavenger of peroxides endogenously generated, whereas Prx1 is an occasional scavenger of peroxides exogenously encountered. Furthermore, genome sequence database search predicted widespread coexistence of the two Prxs among bacteria.

Alpha2-adrenergic receptors in spiral ganglion neurons may mediate protective effects of brimonidine and yohimbine against glutamate and hydrogen peroxide toxicity

Brimonidine, an alpha2-adrenergic receptor (α2-AR) agonist, is thought to be neuroprotective in some types of neurons via the activation of α2-AR. However, it is still unknown whether the α2-ARs exist in cochlear spiral ganglion neurons (SGNs). The authors aimed to demonstrate the presence and localization of α2-ARs in rat-cultured SGNs and to investigate the effect of brimonidine on glutamate- and hydrogen peroxide (H2O2)-induced damage in the primary-cultured rat SGNs. The expression of α2-ARs was determined by reverse transcription-polymerase chain reaction, Western blot analysis and immunofluorescence. Then SGNs were exposed to glutamate or H2O2 respectively with or without brimonidine. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Apoptosis was determined by acridine orange and Hoechst 33342/propidium iodide double staining. The protein expressions of α2-ARs, Bax, Bcl-2, Caspase-9, Caspase-3, p-ERK1/2, iNOS, and artemin were determined by Western blot respectively. The cell viability was markedly reduced after exposure of glutamate (1mM) or H2O2 (300μM) to SGNs. Treatment with brimonidine protected SGNs against glutamate- or H2O2-induced cell damage, enhanced SGNs survival, decreased the elevation of Bax, Caspase-9, Caspase-3, p-ERK1/2, and artemin triggered by glutamate or H2O2, and altered the expressions of Bcl-2 and iNOS. These protective effects of brimonidine can be reversed by yohimbine. Overall, the study describes the localization of α2-ARs in rat-cultured SGNs and indicates that brimonidine, which may work directly via interaction with α2-ARs, attenuates glutamate- and H2O2-induced damage in SGNs by Caspase-dependent modes as well as Caspase-independent modes.

Inducible protection of human astrocytoma 1321N1 cells against hydrogen peroxide and aldehyde toxicity by 7-hydroxycoumarin is associated with the upregulation of aldo-keto reductases

Reactive oxygen species (ROS) and consequent aldehydic lipid peroxidation products have been identified as significant in the progression of neurodegenerative diseases such as Alzheimer's and Parkinson's. Understanding and enhancing endogenous cellular protection against oxidants and aldehydes is therefore of interest in developing strategies to combat these diseases. In this study the role of the aldo-keto reductases AKR7A2 and AKR1C3 in protecting human astrocytoma 1321N1 cells against oxidant and aldehyde toxicity was investigated using siRNA gene silencing. Results show that both enzymes are responsible for part of the intrinsic protection against aldehydes and oxidants. Treating cells with sub-lethal concentrations of oxidant or aldehyde stress or with the natural coumarin 7-hydroxycoumarin (umbelliferone) revealed that endogenous resistance to aldehydes and oxidants can be induced significantly. The basis of the inducible protection by 7-hydroxycoumarin was shown to be associated with induction of the aldo-keto reductases AKR7A2 (1.5-fold) and AKR1C (3-fold), and this inducible protection was sufficient to overcome siRNA silencing of AKR1C3. These results indicate the importance of AKR family members in the detoxication of aldehydes, and also show that the natural phytochemical 7-hydroxycoumarin is a potential therapeutic candidate for neurodegenerative diseases.

Green Tea Polyphenol Protection Against 4-Nitroquinoline 1-Oxide-Induced Bone Marrow Lipid Peroxidation and Genotoxicity in Wistar Rats

4-Nitroquinoline 1-oxide (4-NQO) a potent oral carcinogen, widely used for induction of oral carcinogenesis, has been found to induce lipid peroxidation in vivo and in vitro. Green tea contains a high content of polyphenols, which are potent antioxidants. Thus green tea polyphenols (GTP) might be expected play a protective role against 4-NQO induced lipid peroxidation and bone marrow toxicity. In the present study, a dose of 200 mg of GTP/kg b.wt/day was given orally for a week, simultaneously animals received 0.2 ml of 0.5% 4-NQO in propylene glycol (5 mg/ml) injected intramuscularly for three times/week. Oxidants and antioxidants such as malendialdehyde (MDA) and thiols, glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT) were significantly decreased in 4-NQO induced animals except MDA, and these parameters were brought back to near normalcy on treatment with GTP. The results suggest that GTP treatment offers significant protection against 4-NQO induced lipid peroxidation and bone marrow toxicity and might be a promising potential candidate for prevention of mutations leading to cancer.

Metabolic activity and membrane integrity changes in Microcystis aeruginosa – new findings on hydrogen peroxide toxicity in cyanobacteria

The aim of our study was to investigate the intracellular toxicity mechanisms of the photoactive, potentially anti-cyanobacterial agent hydrogen peroxide (H2O2) in Microcystis aeruginosa, which represents one of the most significant toxin-producing cyanobacterial species in European water bodies. Metabolic activity and cell membrane integrity were evaluated by flow cytometry in cyanobacteria exposed to H2O2 in the dark or light; the relationships between exposure effects and the kinetics of hydrogen peroxide decomposition were studied. In the light (irradiance 140 µmol m−2 s−1), cyanobacteria were exposed to initial H2O2 concentrations of 0.00 (control), 0.75, 2.00, and 4.00 mg l−1 respectively, while in the dark concentrations were ten times higher. Flow cytometry and chlorophyll a fluorescence measurements suggested that hydrogen peroxide exposure elicits an immediate decline of metabolic (esterase) activity, measured as a decrease in fluorescein fluorescence after hydrolysis of fluorescein diacetate (FDA), and immediate changes of chlorophyll a fluorescence parameters, followed later by an increase in the percentage of membrane-compromised (SYTOX Green positive) cells. When the concentration of H2O2 used was lethal (in the two highly exposed treatments in the light), a significant drop in total cell counts was detected, whereas in other treatments no drop was observed during the entire experimental period (72 h). Our study also confirmed that light is one of the critical factors affecting H2O2 decomposition and thus greatly influences its toxicity. Whereas in the light, M. aeruginosa exposed to 0.75 mg l−1 H2O2 recovered after all the H2O2 had decomposed, in the dark H2O2 decomposed relatively slowly and its toxic effects on the cyanobacteria were observed over the whole 72-h period, though without cell lysis in any experimental concentration.

Pseudomonas aeruginosa Thiol Peroxidase Protects against Hydrogen Peroxide Toxicity and Displays Atypical Patterns of Gene Regulation

The Pseudomonas aeruginosa PAO1 thiol peroxidase homolog (Tpx) belongs to a family of enzymes implicated in the removal of toxic peroxides. We have shown the expression of tpx to be highly inducible with redox cycling/superoxide generators and diamide and weakly inducible with organic hydroperoxides and hydrogen peroxide (H(2)O(2)). The PAO1 tpx pattern is unlike the patterns for other peroxide-scavenging genes in P. aeruginosa. Analysis of the tpx promoter reveals the presence of a putative IscR binding site located near the promoter. The tpx expression profiles in PAO1 and the iscR mutant, together with results from gel mobility shift assays showing that purified IscR specifically binds the tpx promoter, support the role of IscR as a transcriptional repressor of tpx that also regulates the oxidant-inducible expression of the gene. Recombinant Tpx has been purified and biochemically characterized. The enzyme catalyzes thioredoxin-dependent peroxidation and can utilize organic hydroperoxides and H(2)O(2) as substrates. The Δtpx mutant demonstrates differential sensitivity to H(2)O(2) only at moderate concentrations (0.5 mM) and not at high (20 mM) concentrations, suggesting a novel protective role of tpx against H(2)O(2) in P. aeruginosa. Altogether, P. aeruginosa tpx is a novel member of the IscR regulon and plays a primary role in protecting the bacteria from submillimolar concentrations of H(2)O(2).