Nitric oxide inhibits topoisomerase II activity and induces resistance to topoisomerase II-poisons in human tumor cells.

Background: Etoposide and doxorubicin, topoisomerase II poisons, are important drugs for the treatment of tumors in the clinic. Topoisomerases contain several free sulfhydryl groups which are important for their activity and are also potential targets for nitric oxide (NO)-induced nitrosation. NO, a physiological signaling molecule nitrosates many cellular proteins, causing altered protein and cellular functions.

Nitric Oxide Down-Regulates Topoisomerase I and Induces Camptothecin Resistance in Human Breast MCF-7 Tumor Cells.

Camptothecin (CPT), a topoisomerase I poison, is an important drug for the treatment of solid tumors in the clinic. Nitric oxide (·NO), a physiological signaling molecule, is involved in many cellular functions, including cell proliferation, survival and death. We have previously shown that ·NO plays a significant role in the detoxification of etoposide (VP-16), a topoisomerase II poison in vitro and in human melanoma cells.

Investigating free radical generation in HepG2 cells using immuno-spin trapping.

Oxidative stress can induce the generation of free radicals, which are believed to play an important role in both physiological and pathological processes and a number of diseases such as cancer. Therefore, it is important to identify chemicals which are capable of inducing oxidative stress. In this study, we evaluated the ability of four environmental chemicals, aniline, nitrosobenzene (NB), N,N-dimethylaniline (DMA) and N,N-dimethyl-4-nitrosoaniline (DMNA), to induce free radicals and cellular damage in the hepatoma cell line HepG2.

Tripping up Trp: Modification of protein tryptophan residues by reactive oxygen species, modes of detection, and biological consequences.

Proteins comprise a majority of the dry weight of a cell, rendering them a major target for oxidative modification. Oxidation of proteins can result in significant alterations in protein molecular mass such as breakage of the polypeptide backbone and/or polymerization of monomers into dimers, multimers, and sometimes insoluble aggregates. Protein oxidation can also result in structural changes to amino acid residue side chains, conversions that have only a modest effect on protein size but can have widespread consequences for protein function.

OKN-007 decreases free radical levels in a preclinical F98 rat glioma model.

Free radicals are associated with glioma tumors. Here, we report on the ability of an anticancer nitrone compound, OKN-007 [Oklahoma Nitrone 007; a disulfonyl derivative of α-phenyl-tert-butyl nitrone (PBN)] to decrease free radical levels in F98 rat gliomas using combined molecular magnetic resonance imaging (mMRI) and immunospin-trapping (IST) methodologies. Free radicals are trapped with the spin-trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), to form DMPO macromolecule radical adducts, and then further tagged by immunospin trapping by an antibody against DMPO adducts.

In vivo targeted molecular magnetic resonance imaging of free radicals in diabetic cardiomyopathy within mice.

Free radicals contribute to the pathogenesis of diabetic cardiomyopathy. We present a method for in vivo observation of free radical events within murine diabetic cardiomyopathy. This study reports on in vivo imaging of protein/lipid radicals using molecular MRI (mMRI) and immuno-spin trapping (IST) in diabetic cardiac muscle.

Free radical generation from an aniline derivative in HepG2 cells: a possible captodative effect.

Xenobiotic metabolism can induce the generation of protein radicals, which are believed to play an important role in the toxicity of chemicals and drugs. It is therefore important to identify chemical structures capable of inducing macromolecular free radical formation in living cells. In this study, we evaluated the ability of four structurally related environmental chemicals, aniline, nitrosobenzene, N,N-dimethylaniline, and N,N-dimethyl-4-nitrosoaniline (DMNA), to induce free radicals and cellular damage in the hepatoma cell line HepG2.

In vivo detection of free radicals in mouse septic encephalopathy using molecular MRI and immuno-spin trapping.

Free radicals are known to play a major role in sepsis. Combined immuno-spin trapping and molecular magnetic resonance imaging (MRI) was used to detect in vivo and in situ levels of free radicals in murine septic encephalopathy after cecal ligation and puncture (CLP). DMPO (5,5-dimethyl pyrroline N-oxide) was injected over 6h after CLP, before administration of an anti-DMPO probe (anti-DMPO antibody bound to albumin-gadolinium-diethylene triamine pentaacetic acid-biotin MRI targeting contrast agent).

Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas.

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent) to trap tumor-associated free radicals.

In vivo detection of free radicals using molecular MRI and immuno-spin trapping in a mouse model for amyotrophic lateral sclerosis.

Free radicals associated with oxidative stress play a major role in amyotrophic lateral sclerosis (ALS). By combining immuno-spin trapping and molecular magnetic resonance imaging, in vivo trapped radical adducts were detected in the spinal cords of SOD1(G93A)-transgenic (Tg) mice, a model for ALS. For this study, the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) was administered (ip) over 5 days before administration (iv) of an anti-DMPO probe (anti-DMPO antibody covalently bound to an albumin-gadolinium-diethylenetriamine pentaacetic acid-biotin MRI contrast agent) to trap free radicals.

Immuno-spin trapping of heme-induced protein radicals: Implications for heme oxygenase-1 induction and heme degradation.

Heme, in the presence of hydrogen peroxide, can act as a peroxidase. Intravascular hemolysis results in a massive release of heme into the plasma in several pathophysiological conditions such as hemolytic anemia, malaria, and sickle cell disease. Heme is known to induce heme oxygenase-1(HO-1) expression, and the extent of induction depends on the ratio of albumin to heme in plasma.

Hypericin-mediated photooxidative damage of α-crystallin in human lens epithelial cells.

St. John's wort (Hypericum perforatum), a perennial herb native to Europe, is widely used for and seems to be effective in treatment of mild to moderate depression. Hypericin, a singlet oxygen-generating photosensitizer that absorbs in both the visible and the UVA range, is considered to be one of the bioactive ingredients of St.

The specific interaction of the photosensitizer methylene blue with acetylcholinesterase provides a model system for studying the molecular consequences of photodynamic therapy.

The photosensitizer, methylene blue (MB), generates singlet oxygen ((1)O2) that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark MB inhibits reversibly, binding being accompanied by a bathochromic shift that can be used to show its displacement by other reversible inhibitors binding to the catalytic 'anionic' subsite (CAS), the peripheral 'anionic' subsite (PAS), or bridging them. Data concerning both reversible and irreversible inhibition are here reviewed.

Development of immunoblotting techniques for DNA radical detection.

Radical damage to DNA has been implicated in cell death, cellular dysfunction, and cancer. A recently developed method for detecting DNA radicals uses the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) to trap radicals. The trapped radicals then decay into stable nitrone adducts detectable with anti-DMPO antibodies and quantifiable by ELISA or dot-blot assay.

Targeted oxidation of Torpedo californica acetylcholinesterase by singlet oxygen: identification of N-formylkynurenine tryptophan derivatives within the active-site gorge of its complex with the photosensitizer methylene blue.

The principal role of AChE (acetylcholinesterase) is termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine. The active site of AChE is near the bottom of a long and narrow gorge lined with aromatic residues. It contains a CAS (catalytic 'anionic' subsite) and a second PAS (peripheral 'anionic' site), the gorge mouth, both of which bind acetylcholine via π-cation interactions, primarily with two conserved tryptophan residues.

Detection of Ras GTPase protein radicals through immuno-spin trapping.

Over the past decade immuno-spin trapping (IST) has been used to detect and identify protein radical sites in numerous heme and metalloproteins. To date, however, the technique has had little application toward nonmetalloproteins. In this study, we demonstrate the successful application of IST in a system free of transition metals and present the first conclusive evidence of (•)NO-mediated protein radical formation in the HRas GTPase.

Phototoxicity of nano titanium dioxides in HaCaT keratinocytes--generation of reactive oxygen species and cell damage.

Nano-sized titanium dioxide (TiO(2)) is among the top five widely used nanomaterials for various applications. In this study, we determine the phototoxicity of TiO(2) nanoparticles (nano-TiO(2)) with different molecular sizes and crystal forms (anatase and rutile) in human skin keratinocytes under UVA irradiation. Our results show that all nano-TiO(2) particles caused phototoxicity, as determined by the MTS assay and by cell membrane damage measured by the lactate dehydrogenase (LDH) assay, both of which were UVA dose- and nano-TiO(2) dose-dependent.

In vivo imaging of immuno-spin trapped radicals with molecular magnetic resonance imaging in a diabetic mouse model.

Oxidative stress plays a major role in diabetes. In vivo levels of membrane-bound radicals (MBRs) in a streptozotocin-induced diabetic mouse model were uniquely detected by combining molecular magnetic resonance imaging (mMRI) and immunotrapping techniques. An anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibody (Ab) covalently bound to an albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent (anti-DMPO probe), and mMRI, were used to detect in vivo levels of DMPO-MBR adducts in kidneys, livers, and lungs of diabetic mice, after DMPO administration.

Immunological detection of N-formylkynurenine in porphyrin-mediated photooxided lens α-crystallin.

Crystallin proteins are responsible for maintaining lens transparency and allowing the lens to focus light undistorted onto the retina. The α-crystallins are the major lens crystallins, and function as both structural proteins and chaperones to protect all lens proteins from damage leading to lens deterioration. Because lens crystallin proteins do not turn over, the damage they accumulate can lead to cataracts, the world's leading cause of blindness.

Myoglobin-H2O2 catalyzes the oxidation of β-ketoacids to α-dicarbonyls: mechanism and implications in ketosis.

Acetoacetate (AA) and 2-methylacetoacetate (MAA) are accumulated in metabolic disorders such as diabetes and isoleucinemia. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by myoglobin (Mb)/H(2)O(2). We propose a chemiluminescent route involving a dioxetanone intermediate whose thermolysis yields triplet α-dicarbonyl species (methylglyoxal and diacetyl).

High-fat diet induces an initial adaptation of mitochondrial bioenergetics in the kidney despite evident oxidative stress and mitochondrial ROS production.

Obesity and metabolic syndrome are associated with an increased risk for several diabetic complications, including diabetic nephropathy and chronic kidney diseases. Oxidative stress and mitochondrial dysfunction are often proposed mechanisms in various organs in obesity models, but limited data are available on the kidney. Here, we fed a lard-based high-fat diet to mice to investigate structural changes, cellular and subcellular oxidative stress and redox status, and mitochondrial biogenesis and function in the kidney.

Site-specific radical formation in DNA induced by Cu(II)-H₂O₂ oxidizing system, using ESR, immuno-spin trapping, LC-MS, and MS/MS.

Oxidative stress-related damage to the DNA macromolecule produces a multitude of lesions that are implicated in mutagenesis, carcinogenesis, reproductive cell death, and aging. Many of these lesions have been studied and characterized by various techniques. Of the techniques that are available, the comet assay, HPLC-EC, GC-MS, HPLC-MS, and especially HPLC-MS/MS remain the most widely used and have provided invaluable information on these lesions.

Oxidative stress induces protein and DNA radical formation in follicular dendritic cells of the germinal center and modulates its cell death patterns in late sepsis.

Profound depletion of follicular dendritic cells (FDCs) is a hallmark of sepsis-like syndrome, but the exact causes of the ensuing cell death are unknown. The cell death-driven depletion contributes to immunoparalysis and is responsible for most of the morbidity and mortality in sepsis. Here we have utilized immuno-spin trapping, a method for detection of free radical formation, to detect oxidative stress-induced protein and DNA radical adducts in FDCs isolated from the spleens of septic mice and from human tonsil-derived HK cells, a subtype of germinal center FDCs, to study their role in FDC depletion.

Protein Radical Formation Resulting from Eosinophil Peroxidase-catalyzed Oxidation of Sulfite.

Eosinophil peroxidase (EPO) is an abundant heme protein in eosinophils that catalyzes the formation of cytotoxic oxidants implicated in asthma, allergic inflammatory disorders, and cancer. It is known that some proteins with peroxidase activity (horseradish peroxidase and prostaglandin hydroperoxidase) can catalyze oxidation of bisulfite (hydrated sulfur dioxide), leading to the formation of sulfur trioxide anion radical ((.)SO(3)(-)).

Partial colocalization of oxidized, N-formylkynurenine-containing proteins in mitochondria and Golgi of keratinocytes.

Proteins are the dominant cellular target for oxidative reactions because they comprise the majority of macromolecules. Posttranslational oxidative protein modifications include fragmentation, aggregation and alteration of specific amino acid residues. The amino acids and amino acid residues most susceptible to oxidative modification are those containing sulfur and those with aromatic rings.