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. METHODS: Here, we have evaluated the roles of NO/NO-derived species in the activity/stability of topo II both in vitro and in human tumor cells, and in the cytotoxicity of topo II-poisons, etoposide and doxorubicin. RESULTS: Treatment of purified topo IIα with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of both the catalytic and relaxation activity in vitro, and decreased etoposide-dependent cleavable complex formation in both human HT-29 colon and MCF-7 breast cancer cells. PPNO treatment also induced significant nitrosation of topo IIα protein in these human tumor cells. These events, taken together, caused a significant resistance to etoposide in both cell lines. However, PPNO had no effect on doxorubicin-induced cleavable complex formation, or doxorubicin cytotoxicity in these cell lines. CONCLUSION: Inhibition of topo II function by NO/NO-derived species induces significant resistance to etoposide, without affecting doxorubicin cytotoxicity in human tumor cells. GENERAL SIGNIFICANCE: As tumors express inducible nitric oxide synthase and generate significant amounts of NO, modulation of topo II functions by NO/NO-derived species could render tumors resistant to certain topo II-poisons in the clinic.

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. ·NO/·NO-derived species are reported to modulate activity of several important cellular proteins. As topoisomerases contain a number of free sulfhydryl groups which may be targets of ·NO/·NO-derived species, we have investigated the roles of ·NO/·NO-derived species in the stability and activity of topo I. Here we show that ·NO/·NO-derived species induces a significant down-regulation of topoisomerase I protein via the ubiquitin/26S proteasome pathway in human colon (HT-29) and breast (MCF-7) cancer cell lines. Importantly, ·NO treatment induced a significant resistance to CPT only in MCF-7 cells. This resistance to CPT did not result from loss of topoisomerase I activity as there were no differences in topoisomerase I-induced DNA cleavage in vitro or in tumor cells, but resulted from the stabilization/induction of bcl2 protein. This up-regulation of bcl2 protein in MCF-7 cells was wtp53 dependent as pifithrine-α, a small molecule inhibitor of wtp53 function, completely reversed CPT resistance, suggesting that wtp53 and bcl2 proteins played important roles in CPT resistance. Because tumors in vivo are heterogeneous and contaminated by infiltrating macrophages, ·NO-induced down-regulation of topoisomerase I protein combined with bcl2 protein stabilization could render certain tumors highly resistant to CPT and drugs derived from it in the clinic.

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. There are a wide range of rate constants for amino acid reactivity, with cysteine, methionine, tyrosine, phenylalanine, and tryptophan having the highest rate constants with commonly encountered biological oxidants. Free tryptophan and tryptophan protein residues react at a diffusion-limited rate with hydroxyl radical and also have high rate constants for reactions with singlet oxygen and ozone. Although oxidation of proteins in general and tryptophan residues specifically can have effects detrimental to the health of cells and organisms, some modifications are neutral, whereas others contribute to the function of the protein in question or may act as a signal that damaged proteins need to be replaced. This review provides a brief overview of the chemical mechanisms by which tryptophan residues become oxidized, presents both the strengths and the weaknesses of some of the techniques used to detect these oxidative interactions, and discusses selected examples of the biological consequences of tryptophan oxidation in proteins from animals, plants, and microbes.

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 this study, we combined mMRI with a biotin-Gd-DTPA-albumin-based contrast agent for signal detection with the specificity of an antibody for DMPO nitrone adducts (anti-DMPO probe), to detect in vivo free radicals in OKN-007-treated rat F98 gliomas. OKN-007 was found to significantly decrease (P < 0.05) free radical levels detected with an anti-DMPO probe in treated animals compared to untreated rats. Immunoelectron microscopy was used with gold-labeled antibiotin to detect the anti-DMPO probe within the plasma membrane of F98 tumor cells from rats administered anti-DMPO in vivo. OKN-007 was also found to decrease nuclear factor erythroid 2-related factor 2, inducible nitric oxide synthase, 3-nitrotyrosine, and malondialdehyde in ex vivo F98 glioma tissues via immunohistochemistry, as well as decrease 3-nitrotyrosine and malondialdehyde adducts in vitro in F98 cells via ELISA. The results indicate that OKN-007 effectively decreases free radicals associated with glioma tumor growth. Furthermore, this method can potentially be applied toward other types of cancers for the in vivo detection of macromolecular free radicals and the assessment of antioxidants.

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. Cytotoxicity was assessed using lactate dehydrogenase assays, and morphological changes were observed using phase contrast microscopy. Protein free radicals were detected by immuno-spin trapping using in-cell western experiments and confocal microscopy to determine the subcellular locale of free radical generation. DMNA induced free radical generation, lactate dehydrogenase release, and morphological changes in HepG2 cells, whereas aniline, nitrosobenzene, N,N-dimethylaniline did not. Confocal microscopy showed that DMNA induced free radical generation mainly in the cytosol. Preincubation of HepG2 cells with N-acetylcysteine and 2,2'-dipyridyl significantly prevented free radical generation on subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide had no effect. These results suggest that DMNA is metabolized to reactive free radicals capable of generating protein radicals which may play a critical role in DMNA toxicity. We propose that the captodative effect, the combined action of the electron-releasing dimethylamine substituent, and the electron-withdrawing nitroso substituent, leads to a thermodynamically stabilized radical, facilitating enhanced protein radical formation by DMNA.

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. Cytotoxicity was assessed using lactate dehydrogenase (LDH) assays and morphological changes were observed using phase contrast microscopy. Free radicals were detected by immuno-spin trapping (IST) in in-cell western experiments or in confocal microscopy experiments to determine the subcellular localization of free radical generation. DMNA induced free radical generation, LDH release and morphological changes in HepG2 cells whereas aniline, NB and DMA did not. Confocal microscopy showed that DMNA induced free radical generation mainly in the cytosol. Preincubation of HepG2 cells with N-acetylcysteine and 2,2'-dipyridyl significantly prevented free radical generation upon subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide did not. These results suggest that DMNA induces oxidative stress and that reactive oxygen species, metals and free radical generation play a critical role in DMNA-induced cytotoxicity.

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. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p<0.001) in MR signal intensity or a significant decrease (p<0.001) in T1 relaxation, measured as %T1 change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p<0.0001) in T1 relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin-Gd-DTPA-biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p<0.001) and 3-nitrotyrosine (3-NT) (p<0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.

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). In vitro assessment of the anti-DMPO probe in oxidatively stressed mouse astrocytes significantly decreased T1 relaxation (p < 0.0001) compared to controls. MRI detected the presence of anti-DMPO adducts via a substantial decrease in %T1 change within the hippocampus, striatum, occipital, and medial cortex brain regions (p < 0.01 for all) in septic animals compared to shams, which was sustained for over 60 min (p < 0.05 for all). Fluorescently labeled streptavidin was used to target the anti-DMPO probe biotin, which was elevated in septic brain, liver, and lungs compared to sham. Ex vivo DMPO adducts (qualitative) and oxidative products, including 4-hydroxynonenal and 3-nitrotyrosine (quantitative, p < 0.05 for both), were elevated in septic brains compared to shams. This is the first study that has reported on the detection of in vivo and in situ levels of free radicals in murine septic encephalopathy.

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. MRI was used to detect the presence of the anti-DMPO radical adducts by a significant sustained increase in MR signal intensities (p < 0.05) or anti-DMPO probe concentrations measured from T1 relaxations (p < 0.01). The biotin moiety of the anti-DMPO probe was targeted with fluorescence-labeled streptavidin to locate the probe in excised tissues. Negative controls included either Tg ALS mice initially administered saline rather than DMPO followed by the anti-DMPO probe or non-Tg mice initially administered DMPO and then the anti-DMPO probe. The anti-DMPO probe was found to bind to neurons via colocalization fluorescence microscopy. DMPO adducts were also confirmed in diseased/nondiseased tissues from animals administered DMPO. Apparent diffusion coefficients from diffusion-weighted images of spinal cords from Tg mice were significantly elevated (p < 0.001) compared to wild-type controls. This is the first report regarding the detection of in vivo trapped radical adducts in an ALS model. This novel, noninvasive, in vivo diagnostic method can be applied to investigate the involvement of free radical mechanisms in ALS rodent models.

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. HO-1 degrades heme and ultimately generates the antioxidant bilirubin. Heme also causes oxidative stress in cells, but whether it causes protein-radical formation has not yet been studied. In the literature, two purposes for the degradation of heme by HO-1 are discussed. One is the production of the antioxidant bilirubin and the other is the prevention of heme-dependent adverse effects. Here we have investigated heme-induced protein-radical formation, which might have pathophysiological consequences, and have used immuno-spin trapping to establish the formation of heme-induced protein radicals in two systems: human serum albumin (HSA)/H2O2 and human plasma/H2O2.We found that excess heme catalyzed the formation of HSA radicals in the presence of hydrogen peroxide. When heme and hydrogen peroxide were added to human plasma, heme was found to oxidize proteins, primarily and predominantly HSA; however, when HSA-depleted plasma was used, heme triggered the oxidation of several other proteins, including transferrin. Thus, HSA in plasma protected other proteins from heme/H2O2-induced oxidation. The antioxidants ascorbate and uric acid significantly attenuated protein-radical formation induced by heme/H2O2; however, bilirubin did not confer significant protection. Based on these findings, we conclude that heme is degraded by HO-1 because it is a catalyst of protein-radical formation and not merely to produce the relatively inefficient antioxidant bilirubin.

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. John's wort, and commercial preparations are frequently calibrated to contain a standard concentration. Hypericin can accumulate in ocular tissues, including lenses, and can bind in vitro to α-crystallin, a major lens protein. α-crystallin is required for lens transparency and also acts as a chaperone to ensure its own integrity and the integrity of all lens proteins. Because there is no crystallin turnover, damage to α-crystallin is cumulative over the lifetime of the lens and can lead to cataracts, the principal cause of blindness worldwide. In this work we study hypericin photosensitization of α-crystallin and detect extensive polymerization of bovine α-crystallin exposed in vitro to hypericin and UVA. We use fluorescence confocal microscopy to visualize binding between hypericin and α-crystallin in a human lens epithelial (HLE) cell line. Further, we show that UVA irradiation of hypericin-treated HLE cells results in a dramatic decrease in α-crystallin detection concurrent with a dramatic accumulation of the tryptophan oxidation product N-formylkynurenine (NFK). Examination of actin in HLE cells indicates that this cytoskeleton protein accumulates NFK resulting from hypericin-mediated photosensitization. This work also shows that filtration of wavelengths <400nm provides incomplete protection against α-crystallin modification and NFK accumulation, suggesting that even by wearing UV-blocking sunglasses, routine users of St. John's wort cannot adequately shield their lenses from hypericin-mediated photosensitized damage.

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. However, the sequences of DNA that are damaged are likely to be as important as the total level of damage. Therefore, we have developed immunoblotting methods for detection of DNA nitrone adducts on electrophoretically separated DNA, comparable to Western blotting for proteins. These new techniques not only allow the assessment of relative radical adduct levels, but can reveal specific DNA fragments, and ultimately nucleotides, as radical targets. Moreover, we have determined that denaturation of samples into single-stranded DNA enhances the detection of DNA-DMPO adducts in our new blotting methods and also in ELISA.

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. MB protects TcAChE from thermal denaturation, and differential scanning calorimetry reveals a ~8 °C increase in the denaturation temperature. The crystal structure of the MB/TcAChE complex reveals a single MB stacked against W279 in the PAS, pointing down the gorge towards the CAS. The intrinsic fluorescence of the irreversibly inhibited enzyme displays new emission bands that can be ascribed to N'-formylkynurenine (NFK); this was indeed confirmed using anti-NFK antibodies. Mass spectroscopy revealed that two Trp residues, Trp84 in the CAS, and Trp279 in the PAS, were the only Trp residues, out of a total of 14, significantly modified by photo-oxidation, both being converted to NFK. In the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Thus, photo-oxidative damage caused by MB involves targeted release of (1)O2 by the bound photosensitizer within the aqueous milieu of the active-site gorge.

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. It was shown previously that generation of (1)O(2) by illumination of MB (Methylene Blue) causes irreversible inactivation of TcAChE (Torpedo californica AChE), and suggested that photo-oxidation of tryptophan residues might be responsible. In the present study, structural modification of the TcAChE tryptophan residues induced by MB-sensitized oxidation was investigated using anti-N-formylkynurenine antibodies and MS. From these analyses, we determined that N-formylkynurenine derivatives were specifically produced from Trp(84) and Trp(279), present at the CAS and PAS respectively. Peptides containing these two oxidized tryptophan residues were not detected when the competitive inhibitors, edrophonium and propidium (which should displace MB from the gorge) were present during illumination, in agreement with their efficient protection against the MB-induced photo-inactivation. Thus the bound MB elicited selective action of (1)O(2) on the tryptophan residues facing on to the water-filled active-site gorge. The findings of the present study thus demonstrate the localized action and high specificity of MB-sensitized photo-oxidation of TcAChE, as well as the value of this enzyme as a model system for studying the mechanism of action and specificity of photosensitizing agents.

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. HRas is a nonmetalloprotein that plays a critical role in regulating cell-growth control. Protein radical formation in Ras GTPases has long been suspected of initiating premature release of bound guanine nucleotide. This action results in altered Ras activity both in vitro and in vivo. As described herein, successful application of IST may provide a means for detecting and identifying radical-mediated Ras activation in many different cancers and disease states in which Ras GTPases play an important role.

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. Magnetic resonance signal intensities, which increase in the presence of a Gd-based molecular probe, were significantly higher within the livers, kidneys, and lungs of diabetic animals administered the anti-DMPO probe compared with controls. Fluorescence images validated the location of the anti-DMPO probe in excised tissues via conjugation of streptavidin-Cy3, which targeted the probe biotin moiety, and immunohistochemistry was used to validate the presence of DMPO adducts in diabetic mouse livers. This is the first report of noninvasively imaging in vivo levels of MBRs within any disease model. This method can be specifically applied toward diabetes models for in vivo assessment of free radical levels, providing an avenue to more fully understand the role of free radicals in diabetes.

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. The smaller the particle size of the nano-TiO(2) the higher the cell damage. The rutile form of nano-TiO(2) showed less phototoxicity than anatase nano-TiO(2). The level of photocytotoxicity and cell membrane damage is mainly dependent on the level of reactive oxygen species (ROS) production. Using polyunsaturated lipids in plasma membranes and human serum albumin as model targets, and employing electron spin resonance (ESR) oximetry and immuno-spin trapping as unique probing methods, we demonstrated that UVA irradiation of nano-TiO(2) can induce significant cell damage, mediated by lipid and protein peroxidation. These overall results suggest that nano-TiO(2) is phototoxic to human skin keratinocytes, and that this phototoxicity is mediated by ROS generated during UVA irradiation.

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. Photosensitizing porphyrins can accumulate in the eye through either endogenous metabolism or through therapeutic or diagnostic procedures. Porphyrin buildup exacerbates lens aging through increased levels of singlet oxygen, resulting in protein polymerization and amino acid residue alteration. Tryptophans oxidize to kynurenine and N-formylkynurenine (NFK) causing irreversible changes in the refractive index of the normally transparent lens, leading to development of cataracts. Additionally, NFK is itself a photosensitizer, and its presence exacerbates lens deterioration. This work uses anti-NFK antiserum to study porphyrin-facilitated photooxidation of α-crystallin tryptophan residues. In vitro experiments show that four biologically interesting porphyrins mediate α-crystallin polymerization and accumulation of both protein radicals and NFK. Confocal microscopy of cultured human lens epithelial cells indicates that while all four porphyrins photosensitize cellular proteins, not all oxidize the tryptophans of cellular α-crystallin to NFK.

Myoglobin-H2O2 catalyzes the oxidation of ß-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). The observed ultraweak chemiluminescence increased linearly on raising the concentration of either Mb (10-500 µM) or AA (10-100 mM). Oxygen uptake studies revealed that MAA is almost a 100-fold more reactive than AA. EPR spin-trapping studies with MNP/MAA revealed the intermediacy of an α-carbon-centered radical and acetyl radical. The latter radical, probably derived from triplet diacetyl, is totally suppressed by sorbate, a well-known quencher of triplet carbonyls. Furthermore, an EPR signal assignable to MNP-AA(•) adduct was observed and confirmed by isotope effects. Oxygen consumption and α-dicarbonyl yield were shown to be dependent on AA or MAA concentrations (1-50 mM) and on H(2)O(2) or tert-butOOH added to the Mb-containing reaction mixtures. That ferrylMb is involved in a peroxidase cycle acting on the substrates is suggested by the reaction pH profiles and immunospin-trapping experiments. The generation of radicals and triplet dicarbonyl products by Mb/H(2)O(2)/ß-ketoacids may contribute to the adverse health effects of ketogenic unbalance.

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. However, accurate measurement of DNA damage has been a matter of debate. In particular, there have been reports of artifactual oxidation leading to erroneously high damage estimates. Further, most of these techniques measure the end product of a sequence of events and thus provide only limited information on the initial radical mechanism. We report here a qualitative measurement of DNA damage induced by a Cu(II)-H2O2 oxidizing system using immuno-spin trapping (IST) with electron paramagnetic resonance (EPR), MS, and MS/MS. The radical generated is trapped by DMPO immediately upon formation. The DMPO adduct formed is initially EPR active but subsequently is oxidized to the stable nitrone, which can then be detected by IST and further characterized by MS and MS/MS.