Eosinophil peroxidase catalyzes bromination of free nucleosides and double-stranded DNA

Chronic parasitic infections are a major risk factor for cancer development in many underdeveloped countries. Oxidative damage of DNA may provide a mechanism linking these processes. Eosinophil recruitment is a hallmark of parasitic infections and many forms of cancer, and eosinophil peroxidase (EPO), a secreted hemoprotein, plays a central role in oxidant production by these cells. However, mechanisms through which EPO may facilitate DNA oxidation have not been fully characterized. Here, we show that EPO effectively uses plasma levels of bromide as a cosubstrate to brominate bases in nucleotides and double-stranded DNA, forming several stable novel brominated adducts. Products were characterized by HPLC with on-line UV spectroscopy and electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS). Ring assignments for brominated purine bases as their 8-bromo adducts were identified by NMR spectroscopy. Using stable isotope dilution LC/ESI/MS/MS, we show that while guanine is the preferred purine targeted for bromination as a free nucleobase, 8-bromoadenine is the major purine oxidation product generated following exposure of double-stranded DNA to either HOBr or the EPO/H(2)O(2)/Br(-) system. Bromination of nucleobases was inhibited by scavengers of hypohalous acids such as the thioether methionine, but not by a large molar excess of primary amines. Subsequently, N-monobromoamines were demonstrated to be effective brominating agents for both free nucleobases and adenine within intact DNA. A rationale for selective modification of adenine, but not guanine, in double-stranded DNA based upon stereochemical criteria is presented. Collectively, these results suggest that specific brominated DNA bases may serve as novel markers for monitoring oxidative damage of DNA and the nucleotide pool by brominating oxidants.     

DNA damage caused by lipid peroxidation products

Lipid peroxidation is a process involving the oxidation of polyunsaturated fatty acids (PUFAs), which are basic components of biological membranes. Reactive electrophilic compounds are formed during lipid peroxidation, mainly alpha, beta-unsaturated aldehydes. These compounds yield a number of adducts with DNA. Among them, propeno and substituted propano adducts of deoxyguanosine with malondialdehyde (MDA), acrolein, crotonaldehyde and etheno adducts, resulting from the reactions of DNA bases with epoxy aldehydes, are a very important group of adducts. The epoxy aldehydes are more reactive towards DNA than the parent unsaturated aldehydes. The compounds resulting from lipid peroxidation mostly react with DNA showing both genotoxic and mutagenic action; among them, 4-hydroxynonenal is the most genotoxic, while MDA is the most mutagenic. DNA damage caused by the adducts of lipid peroxidation products with DNA can be removed by the repairing action of glycosylases. The formed adducts have been hitherto analyzed using the IPPA (Imunopurification-(32)P-postlabelling assay) method and via gas chromatography/electron capture negtive chemical ionization/mass spectrometry (GC/EC NCI/MS). A combination of liquid chromatography with electrospray tandem mass spectrometry (LC/ES-MSMS) with labelled inner standard has mainly been used in recent years.     

Site-specific DNA damage caused by lipid peroxidation products

DNA damage induced by autoxidized lipids was investigated using covalently closed circular (supercoiled) DNA and DNA fragments of defined sequence. DNA-strand-breaking substances accumulated during autoxidation of methyl linolenate, and strand breakage was measured with samples taken at different times. The DNA-strand-breaking activity reached its maximum a little after the peak value of peroxide and decreased upon further autoxidation. The peak of the DNA-strand-breaking activity did not always coincide with the peak of thiobarbituric acid reactants or of conjugated diene, either. The DNA-strand-breaking reaction was dependent on metal ions and was inhibited by potassium iodide and tiron and partially by catalase, suggesting the involvement of radical species and/or oxygen radicals. No direct cleavage of singly end-labeled 100-200 basepair DNA fragments by autoxidized methyl linolenate and cupric ion was detected under the conditions used. Cleavage occurred during subsequent heating in piperidine after the reaction. The alkali-labile damage was preferentially induced at pyrimidine residues, especially in dinucleotide sequences of pyrimidine-guanine (5'----3'), which was determined by sequencing.     

High-performance liquid chromatography determination of 5-methyl-2'-deoxycytidine, 2'-deoxycytidine, and other deoxynucleosides and nucleosides in DNA digests

This work has undertaken liquid chromatographic separation of nucleosides and deoxynucleosides. Two different columns with three mobile phases (A, deionized water; B, 50 mM phosphate buffer (pH 4.0); C, methanol) and slightly different gradient programs were used. The elution order was as follows: cytidine (C), 2′-deoxycytidine (dC), uridine (U), 5-methyl-2′-cytidine (5mC), 5-methyl-2′-deoxycytidine (5mdC), guanosine (G), deoxyguanosine (dG), 2′-deoxythymidine (dT), adenosine (A), and 2′-deoxyadenine (dA). Using a Luna C18 Phenomenex column (150 × 4.6 mm, 5 μm), the separation was performed at 40 °C with a total flow rate of 1 ml/min and a run time of 10 min. The second column was an Agilent C18 (50 × 3 mm, 1.8 μm), for which the run time was 4.5 min with a flow rate of 0.6 ml/min (25 °C). In application to the DNA digests from human THP-1 cells, the quantification of C, dC, U, 5mC, 5mdC, G, dG, and A was performed. The percentages of global methylation were evaluated based on the 5mdC and dC concentrations (c_5mdC / [c_5mdC + c_dC], where c is concentration in μg/ml) and compared with those calculated from the respective peak areas (A_5mdC / [A_5mdC + A_dC], where A is peak area at 254 nm). For peak area measurements, excellent agreement was obtained with the results reported previously in the same cell line. In the quantitative approach, the results of DNA methylation were higher but consistent with the previous data obtained using mass spectrometric detection. Comparing the analytical features of the two procedures, the use of a smaller column could be recommended because it provides efficient separation (capacity factors in the range of 1.29-10.66), a short run time, and feasibility of nucleoside and deoxynucleoside quantification in real-world samples and because it also minimizes the use of reagents.     

Commercial-scale synthesis of protected 2'-deoxycytidine and cytidine nucleosides

Transformation of 2'-deoxyuridine and uridine analogs to protected 2'-deoxycytidine and cytidine analogs has been investigated by two different methods. First, traditional triazolation protocol and second p-nitrophenoxylation method. Our studies conclude that the triazolation method is better and suitable for commercial scale-up.     

Chemistry and biochemistry of lipid peroxidation products

Abstract

Oxidative stress and resulting lipid peroxidation is involved in various and numerous pathological states including inflammation, atherosclerosis, neurodegenerative diseases and cancer. This review is focused on recent advances concerning the formation, metabolism and reactivity towards macromolecules of lipid peroxidation breakdown products, some of which being considered as ‘second messengers’ of oxidative stress. This review relates also new advances regarding apoptosis induction, survival/proliferation processes and autophagy regulated by 4-hydroxynonenal, a major product of omega-6 fatty acid peroxidation, in relationship with detoxication mechanisms. The use of these lipid peroxidation products as oxidative stress/lipid peroxidation biomarkers is also addressed.     

Accumulation of lipid peroxidation products in cataractous lenses

A study was made of the content of lipid peroxidation products (LPP) in the lenses extracted during operations for cataract as well as in transparent human lenses. In a mature cataract, the elevated content of primary, secondary and end products of lipid peroxidation (diene and triene conjugates, Schiff bases) was revealed. The content of LPP was identical in different clinical patterns of a mature cataract (senile, traumatic, complicated), which points to the universal role of lipid peroxidation in lenticular opacity.     

Cataract induction by products of lipid peroxidation

Liposome suspension prepared from the unsaturated phospholipids exposed to lipid peroxidation (LPO) induced posterior subcapsular cataracts after injection into the posterior vitreous of rabbit eyes. In the background of this model lies a type of lens opacity formed during retinal degeneration when toxic peroxide substances diffuse anteriorly through the vitreous body resulting in vitreous opacities and complicated cataracts. Saturated liposomes (prepared from beta-oleoyl-gamma-palmitoyl) L-alpha-lecithin) did not induce lens opacities, which is the evidence that a lipid peroxidation mechanism may be responsible for the posterior cataracts. Along with cataract formation accumulation of LPO fluorescent products in vitreous, aqueous humor and lens was observed. It was followed by a decreased level of reduced glutathione in the lens. The obtained results strongly support the hypothesis of LPO initial role in cataracts.     

A method of identifying lipid peroxidation products in the blood

Newer method of thermofluorescence developed and experimentally used for identification of lipid peroxidation (LPO) products in the blood serum implied the application of 0.02 ml of studied serum onto a paper filter small disk (8mm in diameter) which was placed then on the cuvette of -38 device supplied with a heating element and thermopara. When thermopara was switched on and the blind opened, the cuvette was heated up to 120 degrees C with the heating rate of 60 degrees C per minute. A two-minute recording of fluorescence was simultaneously performed by "K4-003" self-recorder. By measuring the height of the obtained curve peak the intensity of LPO product fluorescence was determined in conditional units (one conditional unit was equal to one cm of curve peak height). A newer method identifying LPO products in the blood serum was more sensitive and time-sparing.     

Isoprostanes and related products of lipid peroxidation in neurodegenerative diseases

Lipid peroxidation is a major outcome of free radical-mediated injury to brain, where it directly damages membranes and generates a number of oxidized products. Some of the chemically and metabolically stable oxidation products are useful in vivo biomarkers of lipid peroxidation. These include the isoprostanes (IsoPs) and isofurans (IsoFs), derived from arachidonic acid (AA), and neuroprostanes (NeuroPs), derived from docosahexaenoic acid (DHA). We have shown increased levels of IsoPs, NeuroPs, and IsoFs in diseased regions of brain from patients who died from advanced Alzheimer's disease (AD) or Parkinson's disease (PD). Increased cerebrospinal fluid (CSF) levels of IsoPs are present in patients with AD or Huntington's disease (HD) early in the course of their illness, and CSF IsoPs may improve the laboratory diagnostic accuracy for AD. In contrast, quantification of IsoPs in plasma and urine of AD patients has yielded inconsistent results. These results indicate that brain lipid peroxidation is a potential therapeutic target early in the course of AD and HD, that CSF IsoPs may aid in the assessment of anti-oxidant experimental therapeutics and laboratory diagnosis of AD.     

Phosphorescent analysis of lipid peroxidation products in liposomes]

It was found that lipid peroxidation products incorporated into liposomes prepared from oxidized preparations of bovine heart phosphatidylcholine and the total lipid fraction of human erythrocyte membranes are able to phosphoresce at room temperature was studied. The temperature dependences of kinetic and spectral parameters of phosphorescence were measured. It is shown that mechanism of phosphorescence quenching of lipid chromophores has a dynamic nature. It is proposed to use endogenic molecules of the lipid peroxidation products capable of phosphorescence as intrinsic phosphorescence probes for studying the slow molecular dynamics of lipids in artificial and biological membranes in a millisecond range.     

Interaction of lipid peroxidation products with nuclear macromolecules

The interaction of lipid peroxidation products with nuclear macromolecules was investigated in rat liver nuclei labelled with [3H]arachidonic acid. Lipid peroxidation reactions were driven both non-enzymatically and enzymatically by the addition of ascorbate-Fe2+ or NADPH-ADP-Fe3+, respectively, to the incubation mixtures. The extent of peroxidation was evaluated by the formation of thiobarbituric acid chromophore and of radioactive hydrophilic peroxidation products. The results obtained show that: (1) nuclear membrane lipid peroxidation products formed during incubation interact with DNA and total nuclear proteins; (2) non-enzymatic lipid peroxidation processes induced a 40% larger association of peroxidation products to DNA compared to processes driven enzymatically, whereas the corresponding interaction with total nuclear proteins was similar in both peroxidation systems; (3) the radioactivity associated with histones decreased during incubation in the presence of ascorbate-Fe2+ or NADPH-ADP-Fe3+, and increased in control samples (no additions); (4) inhibition of lipid peroxidation by the iron chelator Desferrioxamine B prevented the association of peroxidation products to nuclear macromolecules; (5) the levels of radioactivity found in DNA after 180 min of incubation would represent the formation of 0.6-1.0 adducts per 10(6) DNA bases. The results obtained provide evidence for an interaction between lipid peroxidation products and chromatin in the interior of the cell nucleus.     

Methods for determination of aldehydic lipid peroxidation products

A complex pattern of aldehydes (alkanals, 2-alkenals, 2,4-alkadienals, 4-hydroxyalkenals) is generated by peroxidizing biological samples. Several methods based on HPLC or GC-MS have been developed to qualitatively and quantitatively measure the aldehydes in tissues, cells and cell fractions exposed to various pro-oxidative stimuli. 4-Hydroxynonenal, hexanal and propanal are, besides malonaldehyde, the most abundant aldehydes formed. The high sensitivity of the methods also allows the measurement of physiological aldehyde levels in plasma or low density lipoproteins and this could be of great importance for in vivo studies.     

Lipids and lipid peroxidation products in the pathogenesis of age-related macular degeneration

In people over 50, age-related macular degeneration (ARMD) has become the most common cause for severe visual loss and legal blindness in all industrialized nations. Currently, there is no effective treatment for the majority of patients. To develop new and effective modes of therapy, understanding of the molecular basis of the disease in mandatory. However, the pathogenesis of ARMD is still poorly understood. Several lines of evidence suggest that aging changes of the retinal pigment epithelium (RPE), in particular the accumulation of autofluorescent lipofuscin granules in the lysosomal compartment of postmitotic RPE cells, play a key role in the pathogenesis of the disease. Recent studies indicate that lipidic compounds of lipofuscin, represented by the retinoid A2-E, and protein damage by lipid peroxidation products, in particular malondialdehyde and 4-hydroxynonenal, induce lysosomal dysfunction and lipofuscinogenesis in the RPE. The possible mechanisms underlying this lysosomal dysfunction and the resulting adverse effects on overall RPE function are discussed.     

Isoprostanes: novel bioactive products of lipid peroxidation

Isoprostanes, are a novel group of prostaglandin-like compounds that are biosynthesised from esterified polyunsaturated fatty acid (PUFA) through a non-enzymatic free radical-catalysed reaction. Several of these compounds possess potent biological activity, as evidenced mainly through their pulmonary and renal vasoconstrictive effects, and have short half-lives. It has been shown that isoprostanes act as full or partial agonists through thromboxane receptors. Both human and experimental studies have indicated associations of isoprostanes and severe inflammatory conditions, ischemia-reperfusion, diabetes and atherosclerosis. Reports have shown that F₂-isoprostanes are authentic biomarkers of lipid peroxidation and can be used as potential in vivo indicators of oxidant stress in various clinical conditions, as well as in evaluations of antioxidants or drugs for their free radical-scavenging properties.

Higher levels of F₂-isoprostanes have been found in the normal human pregnancy compared to non-pregnancy, but their physiological role has not been well studied so far. Since bioactive F₂-isoprostanes are continuously formed in various tissues and large amounts of these potent compounds are found unmetabolised in their free acid form in the urine in normal basal conditions with a wide inter-individual variation, their role in the regulation of normal physiological functions could be of further biological interest, but has yet to be disclosed. Their potent biological activity has attracted great attention among scientists, since these compounds are found in humans and animals in both physiological and pathological conditions and can be used as reliable biomarkers of lipid peroxidation.