Detection and characterization of hydroxyl radical adducts by mass spectrometry

The study of the influence of free radicals in the biological process depends primarily on the capacity to detect these reactive species. In this work we have studied the application of mass spectrometry to the identification of hydroxyl radical species. The detection and identification by collisional activation mass-analyzed ion kinetic energy spectrometry (CA-MIKES) of a spin adduct of DMPO with the hydroxyl radical [(DMPO + O) + H]+ (m/z 130) has demonstrated that mass spectrometry can be a powerful tool in the detection and identification of spin adducts of DMPO with hydroxyl radical species. We were also able to detect the capture of secondary free radicals using ethanol by detecting and identifying the corresponding adduct [(DMPO + ethanol) + H]+. Other spin adducts have also been detected and identified. We consider that the use of mass spectrometry is a relevant technique for the detection of free hydroxyl radicals, especially in complex mixtures, since mass spectrometry is able to discriminate these adducts in such situations. Moreover, using this approach, it was possible to identify new spin adducts.     

Effect of hydration on the induction of strand breaks and base lesions in plasmid DNA films by gamma-radiation

The yields of gamma-radiation-induced single- and double-strand breaks (ssb's and dsb's) as well as base lesions, which are converted into detectable ssb by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), at 278 K have been measured as a function of the level of hydration of closed-circular plasmid DNA (pUC18) films. The yields of ssb and dsb increase slightly on increasing the level of hydration (Gamma) from vacuum-dried DNA up to DNA containing 15 mol of water per mole of nucleotide. At higher levels of hydration (15 < Gamma < 35), the yields are constant, indicating that H2O*+ or diffusible hydroxyl radicals, if produced in the hydrated layer, do not contribute significantly to the induction of strand breaks. In contrast, the yields of base lesions, recognized by Nth and Fpg, increase with increasing hydration of the DNA over the range studied. The maximum ratios of the yields of base lesions to that of ssb are 1.7:1 and 1.4:1 for Nth- and Fpg-sensitive sites, respectively. The yields of additional dsb, revealed after enzymatic treatment, increase with increasing level of hydration of DNA. The maximum yield of these enzymatically induced dsb is almost the same as that for prompt, radiation-induced dsb's, indicating that certain types of enzymatically revealed, clustered DNA damage, e.g., two or more lesions closely located, one on each DNA strand, are induced in hydrated DNA by radiation. It is proposed that direct energy deposition in the hydration layer of DNA produces H2O*+ and an electron, which react with DNA to produce mainly base lesions but not ssb. The nucleobases are oxidized by H2O*+ in competition with its conversion to hydroxyl radicals, which if formed do not produce ssb's, presumably due to their scavenging by Tris present in the samples. This pathway plays an important role in the induction of base lesions and clustered DNA damage by direct energy deposition in hydrated DNA and is important in understanding the processes that lead to radiation degradation of DNA in cells or biological samples.     

Quantitative Determination of Cyclobutane Thymine Dimers in DNA by Stable Isotope-Dilution Mass Spectrometry

In order to understand the role of UV-induced DNA lesions in biological processes such as mutagenesis and carcinogenesis, it is essential to detect and quantify DNA damage in cells. In this paper we present a novel and both highly selective and sensitive assay using capillary gas chromatography (GC) combined with mass spectrometry (MS) for the detection and accurate quantitation of a major product of UV-induced DNA damage (cissyn cyclobutadithymine). Quantitation of the cyclobutane thymine dimer was achieved by the use of an internal standard in the form of a stable ²H-labeled analogue. Both isotopically labeled and nonlabeled dimers were prepared directly from their corresponding monomers. Each was identified as their trimethylsilyl ether derivative by GC-MS. Calibration plots were obtained for known quantities of both nonlabeled analyte and internal standard. Quantitation of cis-syn cyclobutadithymine was demonstrated in DNA exposed to UVC radiation over a dose range of 0 to 3500 J m^?2. Under the conditions used, the limit of detection was found to be 20–50 fmol on column (equivalent to 0.02–0.05 nmol dimer per mg DNA). The results of the present study indicate that capillary GC-MS is an ideally suited technique for selective and sensitive quantification of cis-syn cyclobutadithymine in DNA and hence UV-induced DNA damage.     

Oxidative DNA Damage in the Photolysis of Af-Hydroxy-2-Pyridone, a Specific Hydroxyl-Radical Source

Abstract The photooxidative DNA damage by iV-hydroxy-2-pyri-done (1) is caused by hydroxyl radicals, as confirmed by electron paramagnetic resonance studies with the spin trap 5,5-dimethylpyrroline JV-oxide. Irradiation of the pyridone 1 at 300 nm induced strand breaks in super-coiled pBR322 DNA, while in calf-thymus DNA and 2'-deoxyguanosine (dG), respectively, 8-oxoguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine were formed. Time-dependent control experiments disclosed that photoprod-ucts of pyridone 1, e.g. 2-pyridone (3), are not responsible for the modification of DNA. Also the photosensitization by the pyridine-2-one chromophore was excluded, because JV-methylpyridine-2-one (2), which cannot generate hydroxyl radicals, was ineffective in the photooxidation of DNA and dG. Thus, the photolysis of pyridone 1 serves as a specific source of hydroxyl radicals for DNA damage, both strand breaks and base modifications.     

Modulation of photo-oxidative DNA damage by cationic surfactant complexation

The natural packaging of DNA in the cell by histones provides a particular environment affecting its sensitivity to oxidative damage. In this work, we used the complexation of DNA by cationic surfactants to modulate the conformation, the dynamics, and the environment of the double helix. Photo-oxidative damage initiated by benzophenone as the photosensitizer on a plasmid DNA complexed by dodecyltrimethylammonium chloride (DTAC), tetradecyltrimethylammonium chloride (TTAC), cetyltrimethyammonium chloride (CTAC) and bromide (CTAB) was detected by agarose gel electrophoresis. By fluorescent titration in the presence of ethidium bromide (EB) and agarose gel electrophoresis, we experimentally confirmed the complexation diagrams with a critical aggregation concentration on DNA matrix (CAC DNA) delimiting two regions of complexation, according to the DNA-phosphate concentration. The study of the photo-oxidative damage shows, for the first time, a direct correlation between the DNA complexation by these surfactants and the efficiency of DNA cleavage, with a maximum corresponding to the CAC DNA for DTAC and CTAC, and to DNA neutralization for CTAC and CTAB. The localization of a photosensitizer having low water solubility, such as benzophenone, inside the hydrophobic domains formed by the surfactant aggregated on DNA, locally increases the photoinduced cleavage by the free radical oxygen species generated. The inefficiency of a water-soluble quencher of hydroxyl radicals, such as mannitol, confirmed this phenomenon. The detection of photo-oxidative damage constitutes a new tool for investigating DNA complexation by cationic surfactants. Moreover, highlighting the drastically increased sensitivity of a complexed DNA to photo-oxidative damage is of crucial importance for the biological use of surfactants as nonviral gene delivery systems.     

Redox active metal-induced oxidative stress in biological systems

A number of studies performed on biological systems have shown that redox-active metals such as iron and copper as well as other transition metals can undergo redox cycling reactions and produce reactive free radicals termed also reactive oxygen species (ROS) or reactive nitrogen species (RNS). The most representative examples of ROS and RNS are the superoxide anion radical and nitric oxide, respectively, both playing a dual role in biological systems. At low/moderate concentrations of ROS and RNS, they can be involved in many physiological roles such as defense against infectious agents, involvement in a number of cellular signaling pathways and other important biological processes. On the other hand, at high concentrations, ROS and RNS can be important mediators of damage to biomolecules involving DNA, membrane lipids, and proteins. One of the most damaging ROS occurring in biological systems is the hydroxyl radical formed via the decomposition of hydrogen peroxide catalyzed by traces of iron, copper and other metals (the Fenton reaction). The hydroxyl radical is known to react with the DNA molecule, forming 8-OH-Guanine adduct, which is a good biomarker of oxidative stress of an organism and a potential biomarker of carcinogenesis. This review discusses the role of iron and copper in uncontrolled formation of ROS leading to various human diseases such as cancer, cardiovascular disease, and neurological disorders (Alzheimer’s disease and Parkinson’s disease). A discussion is devoted to the various protective antioxidant networks against the deleterious action of free radicals. Metal-chelation therapy, which is a modern pharmacotherapy used to chelate redox-active metals and remove toxic metals from living systems to avoid metal poisoning, is also discussed.     

Hydroxyl mechanism of the antimalarial effect of artemisinin and its analogs

Kinetic schemes for the intramolecular oxidation of four artemisinin analogs, which are used as drugs against malaria, were developed. Each stage of the kinetic scheme is characterized by the enthalpy, activation energy, and rate constant calculated using the model of intersecting parabolas. The competition of mono- and bimolecular radical reactions was taken into account when developing the schemes. The hydroperoxide groups are formed as a result of the intramolecular oxidation of these compounds and generate free radicals in the reaction with Fe^II. Among these free radicals, hydroxyl radicals play the key role, since their yield (n _OH) correlates with the antimalarial activity of the peroxide compound. The efficiency of the drug (index IC₅₀) exponentially depends on n _OH and is expressed by the formula IC₅₀(Artemisinin)/IC₅₀(Compound) = 1.54·10⁻⁶exp(3.9n _OH). The elementary reactions resulting in the generation of hydroxyl radicals are considered. It is supposed that DNA of a malaria parasite is the main biological target for hydroxyl radicals.     

Identification of electrical degradation products of 4-chlorophenol in water.

The electrical decomposition of 4-chlorophenol in water was examined with iridium dioxide doped on atitanium electrode. A number of electrical degradation products of 4-chlorophenol, such as hydroquinone and chlorohydroquinone via the addition of hydroxyl radicals, and dichlorophenol through addition of chlorine radical, were observed as major products. Moreover, hydroxylated chlorobiphenylethers, hydroxylated dibenzo-p-dioxin/furans and hydroxylated chlorobiphenyls formed by a dimerization process during the electrolysis process of 4-chlorophenol were also observed. On the other hand, benzoquinone, muconic acid and aldehyde derivatives that were further oxidative products of hydroquinone formed by photocatalysis process, were not observed. The electrical decomposition products of 4-chlorophenol were trimethylsilylated and then identified by gas chromatography-mass spectrometry. The degradation rate of 4-chlorophenol in water by iridium oxide electrode was measured against the electrical process duration. After iridium electrical process for 120 min, about 50% of 4-chlorophenol was converted into a number of products through oxidation processes. On the basis of the identified products, the degradation pathways of 4-chlorophenol under electrolysis process were proposed.     

In situ DNA oxidative damage by electrochemically generated hydroxyl free radicals on a boron-doped diamond electrode

In situ DNA oxidative damage by electrochemically generated hydroxyl free radicals has been directly demonstrated on a boron-doped diamond electrode. The DNA-electrochemical biosensor incorporates immobilized double-stranded DNA (dsDNA) as molecular recognition element on the electrode surface, and measures in situ specific binding processes with dsDNA, as it is a complementary tool for the study of bimolecular interaction mechanisms of compounds binding to DNA and enabling the screening and evaluation of the effect caused to DNA by radicals and health hazardous compounds. Oxidants, particularly reactive oxygen species (ROS), play an important role in dsDNA oxidative damage which is strongly related to mutagenesis, carcinogenesis, autoimmune inflammatory, and neurodegenerative diseases. The hydroxyl radical is considered the main contributing ROS to endogenous oxidation of cellular dsDNA causing double-stranded and single-stranded breaks, free bases, and 8-oxoguanine occurrence. The dsDNA-electrochemical biosensor was used to study the interaction between dsDNA immobilized on a boron-doped diamond electrode surface and in situ electrochemically generate hydroxyl radicals. Non-denaturing agarose gel-electrophoresis of the dsDNA films on the electrode surface after interaction with the electrochemically generated hydroxyl radicals clearly showed the occurrence of in situ dsDNA oxidative damage. The importance of the dsDNA-electrochemical biosensor in the evaluation of the dsDNA-hydroxyl radical interactions is clearly demonstrated.     

气相色谱/氢火焰检测器检测HL-60细胞DNA中氧化损伤产物8-羟基鸟嘌呤的研究

用0．4mmol／LH2O2处理HL－60细胞株24h,采用气相色谱／氢火焰检测器（GC／FID）检测DNA氧化损伤产物8-羟基鸟嘌呤,并用气相色谱质谱仪选择性离子检测（CGC／MS－SIM）对其进一步鉴定。所用方法的平均回收率为81．7％,RSD小于5％。     

Efficient photoinduced DNA damage by coralyne

The photoinduced DNA damage by the berberine derivative coralyne is presented. The irradiation of coralyne in the presence of plasmid DNA namely, pBR322, leads to remarkably fast DNA damage by single-strand cleavage, as determined by agarose-gel electrophoresis. Even upon exposure to sunlight, almost all of the supercoiled plasmid is converted to the open circular form in less than a minute [c(pBR322) = 3.5 x 10(-9) M; c(coralyne) = 4.3 x 10(-5) M]. The efficiency of the DNA strand cleavage is not decreased in the presence of radical-trapping reagents such as tert-butanol or DMSO. Moreover, the extent of the DNA damage is the same under aerobic conditions and at reduced oxygen concentration. Thus, the formation of reactive intermediates such as hydroxyl radicals or singlet oxygen is excluded. These results show that the exposure of coralyne and derivatives thereof to light, even with moderate light intensity, needs to be avoided during experiments in which their biological activity is assessed by plasmid unwinding assays.     

新型仿生光催化剂HMS-FePcS催化孔雀绿在可见光下的降解历程

在可见光照射下,采用合成的新型仿生光催化剂HMS-FePcS催化降解孔雀绿模拟染料废水.由不同反应时间段反应液的HPLC谱和MS谱中产物峰的变化可知,孔雀绿的催化降解过程为先脱色后矿化.采用固体萃取法对孔雀绿降解的中间产物进行了富集和分离,并使用GC-MS对所得的中间产物进行了鉴定,确定出10余种中间产物.在此基础上对孔雀绿的降解历程进行了推测,指出孔雀绿的光催化降解主要从中心碳原子与二甲氨基苯基之间的C-C键处断裂,4-二甲氨基苯甲酮是此反应最常见的中间产物.当4-二甲氨基苯甲酮被羟基自由基进攻时,生成苯甲酸、对二甲氨基苯甲酸及对二甲氨基苯酚等化合物.这些小分子芳香类中间产物进一步发生羟基化反应,开环生成小分子脂肪酸类化合物.     

PHOTOSENSITIZED DEGRADATION OF DNA BY DAUNOMYCIN

Abstract Visible irradiation of DNA-daunomycin solutions resulted in a decrease of viscosity of the DNA and an increase of the rate of denaturation of DNA in formaldehyde. These changes are consistent with the induction of single-strand breaks in the DNA, some of which pair to cause fragmentation of the DNA. The DNA damage increases with drug: nucleotide ratio up to 0.2 and is diminished beyond that range. The damage also increases with ionic strength up to 0.6 M and is diminished above that value. These results suggest that the non-intercalated form of the drug is involved in the photosensitization process. Radicals that are produced accompanying the degradation have been trapped by 5,5-dimeth-yl-l-pyrroline-1-oxide and identified as hydroxyl radicals from their ESR spectrum. The DNA photosensitized damage is completely inhibited when hydroxyl radicals are removed by the spin-trap, suggesting a direct role for the hydroxyl radicals in the DNA photosensitized degradation process. The implications of the photosensitized DNA damage and the production of hydroxyl radicals in this process are discussed with respect to the medical uses and chemotherapeutic role of daunomycin.     

The Identification of New Triterpenoids in Eucalyptus globulus Wood

Eight polyhydroxy triterpenoid acids, hederagenin, (4α)-23-hydroxybetulinic acid, maslinic acid, corosolic acid, arjunolic acid, asiatic acid, caulophyllogenin, and madecassic acid, with 2, 3, and 4 hydroxyl substituents, were identified and quantified in the dichloromethane extract of Eucalyptus globulus wood by comparing their GC-retention time and mass spectra with standards. Two other triterpenoid acids were tentatively identified by analyzing their mass spectra, as (2α)-2-hydroxybetulinic acid and (2α,4α)-2,23-dihydroxybetulinic acid, with 2 and 3 hydroxyl substituents. Two MS detectors were used, a quadrupole ion trap (QIT) and a quadrupole mass filter (QMF). The EI fragmentation pattern of the trimethylsilylated polyhydroxy structures of these triterpenoid acids is characterized by the sequential loss of the trimethylsilylated hydroxyl groups, most of them by the retro-Diels-Alder (rDA) opening of the C ring with a π-bond at C12-C13. The rDA C-ring opening produces ions at m/z 320 (or 318) and m/z 278 (or 277, 276, 366). Sequential losses of the hydroxyl groups produce ions with m/z from [M - 90] to [M - 90*y], where y is the number of hydroxyl substituents present (from 2 to 4). Moreover, specific cleavage in ring E was observed, passing from m/z 203 to m/z 133 and conducting other major fragments such as m/z 189.     

Purine 5',8-cyclonucleoside lesions: chemistry and biology

5',8-Cyclo-2'-deoxyadenosine and 5',8-cyclo-2'-deoxyguanosine in their 5'R and 5'S diastereomeric forms are tandem-type lesions observed among the DNA modifications and identified in mammalian cellular DNA in vivo. These lesions result from the chemistry of the C5' radicals generated by the attack of HO˙ radicals to 2-deoxyribose units. Quantitative determination of these lesions in biological samples as biomarkers of free radical damage is a challenge. Results reported for irradiated samples of calf thymus DNA have been critically reviewed, underlining the need of further research for the potential involvement of these lesions in human health (76 references).     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS–XII. SPIN TRAPPING STUDY OF THE FREE RADICALS GENERATED DURING THE PHOTOLYSIS OF PHOTO ALLERGENS BITHIONOL and FENTICHLOR

Free radicals were trapped and observed by ESR when photoallergens bithionol and fentichlor were irradiated in the presence of spin traps N- t -butyl-α-phenylnitrone (PBN) and 5,5-dimethyl-pyrroline-N-oxide (DMPO). In the absence of air, both PBN and DMPO trapped a carbon-centered radical. The carbon-centered radical, which was capable of abstracting a hydrogen atom from cysteine, glutathione, ethanol and formate, was identified as an aryl radical derived from the homolytic cleavage of the carbon-chlorine bond. In the presence of air, both carbon-centered radicals and hydroxyl radicals were trapped by DMPO. Under similar conditions, the yield of the hydroxyl radicals was greater from bithionol than from fentichlor. The presence of the hydroxyl radical was confirmed by kinetic experiments employing hydroxyl radical scavengers (ethanol, formate). Superoxide and H₂O₂ were not involved. Experiments with oxygen-¹⁷O indicated that the hydroxyl radicals came exclusively from dissolved oxygen. The precursor of the hydroxyl radical is postulated to be a peroxy intermediate (ArOO*) derived from the reaction of an aryl radical (Ar*) with molecular oxygen. Both bithionol and fentichlor photoionized only when excited in the UVC (<270 nm) region. Free radicals have long been postulated in the photodechlorination of bithionol and fentichlor and the present study provides supporting evidence for such a mechanism. Aryl and hydroxyl radicals are reactive chemical species which may trigger a series of events that culminate in photoallergy.     

The microenvironment effect on the generation of reactive oxygen species by Pd-bacteriopheophorbide

Generation of reactive oxygen species (ROS) is the hallmark of important biological processes and photodynamic therapy (PDT), where ROS production results from in situ illumination of certain dyes. Here we test the hypothesis that the yield, fate, and efficacy of the species evolved highly depend on the dye's environment. We show that Pd-bacteriopheophorbide (Pd-Bpheid), a useful reagent for vascular targeted PDT (VTP) of solid tumors, which has recently entered into phase II clinical trials under the code name WST09 (trade name TOOKAD), forms appreciable amounts of hydroxyl radicals, superoxide radicals, and probably hydrogen peroxide in aqueous medium but not in organic solvents where singlet oxygen almost exclusively forms. Evidence is provided by pico- and nanosecond time-resolved spectroscopies, ESR spectroscopy with spin-traps, time-resolved singlet oxygen phosphorescence, and chemical product analysis. The quantum yield for singlet oxygen formation falls from approximately 1 in organic solvents to approximately 0.5 in membrane-like systems (micelles or liposomes), where superoxide and hydroxyl radicals form at a minimal quantum yield of 0.1%. Analysis of photochemical products suggests that the formation of oxygen radicals involves both electron and proton transfer from (3)Pd-Bpheid at the membrane/water interface to a colliding oxygen molecule, consequently forming superoxide, then hydrogen peroxide, and finally hydroxyl radicals, with no need for metal catalysis. The ability of bacteriochlorophyll (Bchl) derivatives to form such radicals upon excitation at the near infrared (NIR) domain opens new avenues in PDT and research of redox regulation in animals and plants.     

THE GENERATION OF HYDROXYL RADICALS IN BIOLOGIC SYSTEMS: TOXICOLOGICAL ASPECTS

Abstract— The formation of hydroxyl radicals in vitro was studied through their reaction with 2-keto-4-thiomethylbutyric acid to form ethylene gas. The autoxidation reaction of 6-aminodopamine served as a model source of hydroxyl radicals. Ethylene production was suppressed by catalase and by superoxide dismutase, indicating that both hydrogen peroxide and superoxide were involved in the reaction. Hydroxyl radical scavengers (thiourea > benzoate > ethanol) suppressed ethylene production in good agreement with their respective rate constants for reaction with hydroxyl radicals. Urea served as a negative control. Several substituted thiourea derivatives also suppressed ethylene production to a similar degree as thiourea itself. Biologic studies centered on several cytotoxic agents whose mechanisms of action are thought to involve hydroxyl radicals. These agents included alloxan, which destroys the beta cells of the pancreas, and 6-hydroxy- and 6-aminodopamine, which destroy sympathetic nerves. Damage to tissues in vivo was blocked to varying degrees by pretreatment of animals with hydroxyl radical scavengers such as ethanol or the thiourea derivatives. In addition, hydroxyl radical scavengers blocked the action of 5,7-dihydroxytryptamine, a neurotoxin whose effects on noradrenaline neurons were previously shown to be blocked by inhibitors of monoamine oxidase. The data indicate that these cell toxins produce their damaging actions on specific target cells through the intracellular generation of hydroxyl radicals.     

Synthesis,Crystal Structure,Antioxidation and DNA‐Binding Studies of a Zinc(II) Complex with the Bis(N‐allylbenzimidazol‐2‐ylmethyl)aniline

A complex of zinc(II) picrate (pic) with bis(N‐allylbenzimidazol‐2‐ylmethyl)aniline (abba), with composition [Zn(abba)₂](pic)₂, was synthesized and characterized by elemental analysis, electrical conductivity, IR and UV/Vis spectral measurements. The crystal structure of the zinc(II) complex has been determined by single‐crystal X‐ray diffraction. The Zn(II) is bonded to two abba ligands through four benzimidazole nitrogen, resulting in a distorted tetrahedron geometry. The DNA‐binding properties of the ligand and the zinc(II) complex were investigated by electronic absorption, fluorescence spectra and viscosity measurements. The experimental results suggest that the zinc(II) complex binds to DNA in an intercalation mode. In addition, the ligand abba and Zn(II) complex have scavenging effects for hydroxyl radicals and the complex shows stronger scavenging effects for hydroxyl radicals than the ligand.     

Photodynamic properties of dipeptide-modified hypocrellin B derivatives: the role of tyrosine and tryptophan groups

Three long-wavelength absorbing dipeptide-modified hypocrellin B (HB) derivatives, Gly-HB, Tyr-HB, and Trp-HB, were prepared for application in photodynamic therapy (PDT). Their abilities to produce free radicals and singlet oxygen were compared in detail with EPR technique, and their binding interactions with calf thymus DNA (CT DNA) were studied by absorption spectra and DNA melting temperature measurements. Tyr-HB and Trp-HB distinguish themselves from Gly-HB and HB remarkably by their significantly improved efficiencies to generate semiquinone anion radicals, superoxide anion radicals, and hydroxyl radicals, as well as their affinity to CT DNA, as the result of the electron-donating properties and intercalating abilities of tyrosine and tryptophan groups. Tyr-HB and Trp-HB show remarkably enhanced photodamage capabilities on CT DNA than their parent HB in aerobic conditions. Moreover, they possess moderate photodamage abilities on CT DNA even in anaerobic conditions, indicating the role of Type I mechanism in their photodynamic behaviors.