Bactericidal antibiotics and oxidative stress: a radical proposal.

A recent publication suggests that many antibiotics exert their bactericidal effects via the production of hydroxyl radicals, regardless of their molecular targets. This proposal represents an abrupt departure from conventional models, and it will attract further experimental tests.     

Ophthalmic acid is a marker of oxidative stress in plants as in animals

Background

Ophthalmic acid (OPH), γ-glutamyl-L-2-aminobutyryl-glycine, a tripeptide analogue of glutathione (GSH), has recently captured considerable attention as a biomarker of oxidative stress in animals. The OPH and GSH biosynthesis, as well as some biochemical behaviors, are very similar. Here, we sought to investigate the presence of OPH in plants and its possible relationship with GSH, known to possess multiple functions in the plant development, growth and response to environmental changes.

Physiological and biochemical responses to acute ozone-induced oxidative stress in Medicago truncatula.

Oxidative signaling mediated by reactive oxygen species (ROS) is a central component of biotic and abiotic stresses in plants. Acute ozone (O(3)) fumigation is a useful non-invasive treatment for eliciting endogenous ROS in planta. In this study, 38 different accessions of the model legume, Medicago truncatula, from various geographical regions were fumigated with 300 nmol mol(-1) of O(3) for a period of six hours. Phenotypic symptoms were evaluated 24 and 48 h after the end of treatment. A majority of the accessions showed distinct visible damage. Eight accessions showing varying sensitivities to ozone were subjected to biochemical analysis to evaluate correlations between ozone damage and levels of ROS, antioxidants, and lipid peroxidation. Two-way analysis of variance indicated highly significant interactions between O(3) damage and levels of ROS, ascorbate, glutathione and lipid peroxidation. There were significant differences among the accessions for these traits before and after the end of O(3) fumigation, as indicated by equal variance Student's t-test. This study suggests that multiple physiological and biochemical mechanisms may govern O(3) tolerance or sensitivity. Surveying a large collection of germplasm led to identification of multiple resistant and sensitive lines for investigating molecular basis of O(3) phytotoxicity. The most resistant JE154 accession also showed enhanced tolerance to chronic O(3) and dehydration stress, suggesting germplasm with increased tolerance to acute O(3) can be a useful resource for improving resistance to multiple abiotic stressors.     

Hydroxyl radical scavenging activity of beta-N-oxalyl-L-alpha, beta-diaminopropionic acid.

Exogenous hydroxyl radical treatments increased the levels of beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (ODAP) in grass pea seedlings. Lipid peroxidation initiated by hydroxyl radicals can be alleviated when grass pea seedlings were pretreated with exogenous ODAP, suggesting that ODAP might act as an hydroxyl radical scavenger in vivo. Competing with salicylate for hydroxyl radicals in the hydroxyl radical generating/detecting system, ODAP's in vitro hydroxyl radicals scavenging activity was assessed to confirm this result, and the role of ODAP as an effective hydroxyl radical scavenger is discussed.     

Hydroxyl radical production during oxidative deposition of iron in ferritin

The chemistry of oxidative deposition of iron(III) in ferritin and apoferritin is poorly understood. This study was undertaken to look for radicals formed as the hydrous ferric oxide core is developed from Fe(II) and O2. Radicals were observed indirectly by using the spin-trapping reagent N-tert-butyl-alpha-phenylnitrone (PBN) at room temperature and directly by measuring ESR spectra of frozen solutions at 77 K. In both instances, radical production was inhibited by the hydroxyl radical scavenging agents dimethyl sulfoxide, thiourea, and mannitol and enhanced by the addition of hydrogen peroxide. These findings strongly suggest that hydroxyl radical, produced from the iron-catalyzed Haber-Weiss reaction, is a by-product of core formation in ferritin and is a precursor to the observed radicals. The yield of ESR-observable and spin-trapped radicals is quite low, being at the micromolar level when millimolar concentrations of ferrous ion are employed. Furthermore, radical production appears to be confined to the interior of the ferritin molecule, where cellular components would be protected from the oxygen-derived toxic effects of iron. It is postulated that hydroxyl radical-medicated oxidative damage to the protein, a process that may contribute to the formation of hemosiderin from ferritin, leads to the observed radicals. By serving as a sink for hydroxyl radical, the protein shell may therefore efficiently minimize damage to other biomolecules in the cell.     

Free radical scavenging reactions of sulfasalazine, 5-aminosalicylic acid and sulfapyridine: mechanistic aspects and antioxidant activity

Reactions of sulfasalazine (SAZ) and its metabolites, 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP), with various oxidizing and reducing free radicals (hydroxyl, haloperoxyl, one-electron oxidizing, lipid peroxyl, glutathiyl, superoxide, tryptophanyl, etc.) have been studied to understand the mechanistic aspects of its action against free radicals produced during inflammation. Nanosecond pulse radiolysis technique coupled with transient spectrophotometry has been used for in situ generation of free radicals and to follow their reaction pathways. The transients produced in these reactions have been assigned and radical scavenging rate constants have been measured. In addition to scavenging of various primary and secondary free radicals by SAZ, 5-ASA and SP, 5-ASA has also been observed to efficiently scavenge radicals of biomolecules. 5-ASA has been found to be the active moiety of SAZ involved in the scavenging of oxidizing free radicals whereas reduction of SAZ produced molecular radical anion. The study suggests that free radical scavenging activity of 5-ASA may be a major path of pharmacological action of SAZ against inflammatory bowel diseases (IBD).     

An exogenous marker: a novel approach for the characterization of oxidative stress

Oxidative stress (OS) and its consequences which promote alterations in biomolecules, to tissue damage and to the development of pathological conditions, continue to attract many investigators. The identification of reliable biomarker is essential for the characterization of OS and possibly for early discovery of OS-associated diseases. The aim of the present study was to offer a new concept in the development of novel probes for OS, based on the design, synthesis, and utilization of exogenous markers, as alternative to the search for endogenous markers. This article describes: (a) the synthesis of such a marker, linoleoyl tyrosine 2-deoxyguanosyl ester (LTG), constructed from three endogenous subunits: linoleic acid, tyrosine, and 2'-deoxyguanosine, representing the three major groups from which the body is composed, unsaturated fatty acids (USFA), proteins, and DNA, respectively, all bound covalently and (b) the development of analytical tools (LC/MS/MS) to enable the identification of the different LTG oxidized products formed under OS by exposure of LTG to different reactive oxygen species (ROS) such as, copper ions and hypochlorous acid.     

Hydroxyl radical scavenging and antipsoriatic activity of benzoic acid derivatives

The hydroxyl radical scavenging and antipsoriatic activity of a number of lipophilic and hydrophilic benzoic acid derivatives was investigated. To quantify antioxidative effects, a newly introduced test system based on the diminution of the ESR signal of DMPO-OH (generated by Fenton's reagent) by the tested compounds was applied. It was found that the in vitro antioxidative (toward hydroxyl radical) activity of benzoic acid esters decreases with increasing chain length whereas the antipsoriatic activity increases. This effect is discussed in terms of a larger lipophilicity of long-chain esters. Propyl gallate was found to be the most active OH scavenger since it is some orders of magnitude more efficient than "model" antioxidants like alpha-tocopherol or mannitol. The highest antipsoriatic activity was exhibited by hydroxy benzoic acid decyl ester.     

Quantification of noncyclooxygenase derived prostanoids as a marker of oxidative stress

Recently, we discovered there is a unique class of prostaglandin F2-like compounds that are formed in vitro from arachidonoyl-containing lipids in plasma by a free radical-catalyzed mechanism. More recent studies have elucidated that these prostanoids are also produced in vivo in humans by a similar noncyclooxygenase mechanism. Levels of these PGF2 compounds detected by a mass spectrometric assay in normal human plasma and urine range from approximately 5-50 pg/mL and 500-3000 pg/mg creatinine, respectively. Circulating levels of the compounds were shown to increase by as much as 200-fold in animal models of free radical-induced lipid peroxidation. These results suggest that quantification of these prostanoids may provide a new approach to assess oxidative stress in vivo in humans. Potential advantages of this approach are that the mass spectrometric assay has a high degree of sensitivity, accuracy, and specificity and the assay can be used to quantitate these compounds in a variety of biological fluids. In addition, quantification of these compounds is of interest because these compounds possess biological activity. Disadvantages of the assay are the potential of ex vivo formation of these compounds in biological fluids containing lipids and, further, these compounds must be differentiated from PGF2 compounds that are formed via the cyclooxygenase enzyme. In addition, because the levels of these compounds in normal human plasma and urine are relatively high, assaying these compounds in circulating plasma and urine may be somewhat insensitive for the detection of increased production at isolated sites of oxidant injury within the body, in which case sampling near localized sites of their formation may be required.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and validation of a HPLC-ESI-MS/MS method for the determination of 5-aminosalicylic acid and its major metabolite N-acetyl-5-aminosalicylic acid in human plasma

A new HPLC method for the determination of 5-aminosalicylic acid (5-ASA) and N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA) in human plasma was developed and validated. Plasma samples were analyzed after protein precipitation with methanol and the two analytes were separated using a C18 column with a mobile phase composed of 17.5mmol/L acetic acid (pH 3.3):acetonitrile=85:15 (v/v) at 0.2mL/min flow rate. 4-ASA and N-Ac-4-ASA were used as internal standards. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in negative ionization mode and in multiple reaction monitoring acquisition (m/z 152-->108 for 5-ASA; m/z 194-->150 and 194-->107 for N-Ac-5-ASA). The limit of quantification (LOQ) was 50ng/mL for both analytes (0.2ng injected) and matrix-matched standard curves showed linearity up to 4000ng/mL. In the entire analytical range the within- and between-batch precision (R.S.D.%) values were respectively 90% for 5-ASA and >95% for N-Ac-5-ASA (R.S.D.%相似文献   

The relationship between uric acid and its oxidative product allantoin: a potential indicator for the evaluation of oxidative stress in birds

Uric acid is the main nitrogenous waste product in birds but it is also known to be a potent antioxidant. Hominoid primates and birds lack the enzyme urate oxidase, which oxidizes uric acid to allantoin. Consequently, the presence of allantoin in their plasma results from non-enzymatic oxidation. In humans, the allantoin to uric acid ratio in plasma increases during oxidative stress, thus this ratio has been suggested to be an in vivo marker for oxidative stress in humans. We measured the concentrations of uric acid and allantoin in the plasma and ureteral urine of white-crowned sparrows (Zonotrichia leucophrys gambelii) at rest, immediately after 30 min of exercise in a hop/hover wheel, and after 1 h of recovery. The plasma allantoin concentration and the allantoin to uric acid ratio did not increase during exercise but we found a positive relationship between the concentrations of uric acid and allantoin in the plasma and in the ureteral urine in the three activity phases. In the plasma, the slope of the regression describing the above positive relationships was significantly higher immediately after activity. We suggest that the slope indicates the rate of uric acid oxidation and that during activity this rate increases as a result of higher production of free radicals. The present study demonstrates that allantoin is present in the plasma and in the ureteral urine of white-crowned sparrows and therefore might be useful as an indicator of oxidative stress in birds.     

Hydroxyl radical formation and iron-binding proteins. Stimulation by the purple acid phosphatases

The effect of the purple acid phosphatases with binuclear iron centers (uteroferrin and bovine spleen phosphatase) on hydroxyl radical formation by iron-catalyzed Haber-Weiss-Fenton chemistry has been compared to that of lactoferrin and transferrin. Using 5,5-dimethyl-1-pyrroline-1-oxide to detect superoxide and hydroxyl radicals and the xanthine-xanthine oxidase system to generate superoxide and hydrogen peroxide, we have observed by ESR spectroscopy that both phosphatases were able to promote hydroxyl radical formation. Lactoferrin and transferrin were found incapable of giving rise to these reactive species. This can be explained by the fact that lactoferrin and transferrin carry two Fe(III) atoms per molecule, neither of which are readily reduced by biological reductants. In contrast, the phosphatases possess a binuclear iron center in which one of the iron atoms is stabilized in the ferric state, but the other freely undergoes one-electron redox reactions. The redox-active iron may act as a catalyst of the Haber-Weiss-Fenton sequence, thus enabling the reactions generating hydroxyl radical to proceed. The iron complex of diethylenetriamine penta-acetic acid, also redox active, was investigated and found as well to promote Haber-Weiss-Fenton chemistry.     

Detection of lipid peroxidation in vivo: total hydroxyoctadecadienoic acid and 7-hydroxycholesterol as oxidative stress marker

It is important to assess the oxidative injury in vivo accurately and inclusively, as the oxidative stress induced by various oxidants in a random and destructive fashion is considered to play an important role in the pathophysiology of a number of human disorders and diseases. We have developed an improved method for the measurement of lipid peroxidation in vivo, where total hydroxyoctadecadienoic acids (HODE) and 7-hydroxycholesterol (FCOH) were determined by GC/MS analysis from physiological samples after reduction with sodium borohydride and saponification by potassium hydroxide. In this method, both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleate and cholesterol are measured as HODE and FCOH, respectively. The ratio of stereo-isomers, (E,E)-HODE/(E,Z)-HODE, could be also measured. The plasma concentrations of total HODE were obtained as 76.5, 666 and 2225 nM for human, rat and mouse, respectively. It was found that HODE and FCOH could be measured satisfactorily by the present method from plasma, erythrocyte and urine of humans and experimental animals. It was also found that HODE in urine arose from both free and ester forms, while 8-iso-prostaglandin F2alpha was present primarily as a free acid form. As the concentrations of HODE were much higher than 8-iso-prostaglandin F2alpha, HODE may well be used as a good oxidative marker in vivo.     

Ncb5or deficiency increases fatty acid catabolism and oxidative stress

The endoplasmic reticulum-associated NADH cytochrome b(5) oxidoreductase (Ncb5or) is widely distributed in animal tissues. Ncb5or(-/-) mice develop diabetes at age 7 weeks and have increased susceptibility to the diabetogenic oxidant streptozotocin. Ncb5or deficiency also results in lipoatrophy and increased hepatocyte sensitivity to cytotoxic effects of saturated fatty acids. Here we investigate the mechanisms of these phenomena in prediabetic Ncb5or(-/-) mice and find that, despite increased rates of fatty acid uptake and synthesis and higher stearoyl-CoA desaturase (SCD) expression, Ncb5or(-/-) liver accumulates less triacylglycerol (TAG) than wild type (WT). Increased fatty acid catabolism and oxidative stress are evident in Ncb5or(-/-) hepatocytes and reflect increased mitochondrial content, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) expression, fatty acid oxidation rates, oxidative stress response gene expression, and oxidized glutathione content. Ncb5or(-/-) hepatocytes readily incorporate exogenous fatty acids into TAG but accumulate more free fatty acids (FFA) and have greater palmitate-induced oxidative stress responses and cell death than WT, all of which are alleviated by co-incubation with oleate via TAG channeling. A high fat diet rich in palmitate and oleate stimulates both lipogenesis and fatty acid catabolism in Ncb5or(-/-) liver, resulting in TAG levels similar to WT but increased intracellular FFA accumulation. Hepatic SCD-specific activity is lower in Ncb5or(-/-) than in WT mice, although Ncb5or(-/-) liver has a greater increase in Scd1 mRNA and protein levels. Together, these findings suggest that increased FFA accumulation and catabolism and oxidative stress are major consequences of Ncb5or deficiency in liver.     

3,4-Methylenedioxymethamphetamine (MDMA) abuse may cause oxidative stress and potential free radical damage

OBJECTIVE: To investigate whether 3,4-methylenedioxymethamphetamine abuse (MDMA abuse) may cause oxidative stress and potential free radical damage in the bodies of MDMA abusers (MA), and to explore the mechanisms by which MDMA abuse may be causing oxidative stress. METHODS: One hundred and twenty MA and 120 healthy volunteers (HV) were enrolled in a random control study design, in which the level of lipoperoxide (LPO) in erythrocytes, and the levels of Vitamin C (VC), Vitamin E (VE) and beta-carotene (beta-CAR) in plasma as well as the activities of superoxide dismutase (SOD) and catalase (CAT) in erythrocytes were determined by spectrophotometric methods. RESULTS: Compared with the average values of the above biochemical parameters in the HV group, the average value of LPO in erythrocytes in the MA group was significantly increased (P < 0.0001), while the average values of VC, VE and beta-CAR in plasma as well as those of SOD and CAT in erythrocytes in the MA group were significantly decreased (P < 0.0001). The analysis of bivariate correlations suggested that with the increase of the MDMA abuse dose and the MDMA abuse duration, the level of LPO in erythrocytes in the MA was increased (P < 0.0001), while the levels of VC, VE and beta-CAR in plasma as well as the activities of SOD and CAT in erythrocytes in the MA were decreased (P < 0.0001). CONCLUSION: The findings in this study suggest that MDMA abuse may cause oxidative stress and potential free radical damage to MA.     

Oxidized proteins as a marker of oxidative stress during coronary heart surgery

The measurement of the degree of oxidative stress in patients often causes problems because of the lack of useful parameters. Therefore, we used an ELISA technique to evaluate serum protein carbonyls as a parameter of oxidative stress in patients during coronary heart surgery. Protein carbonyls were detected in serum samples of 14 patients undergoing coronary surgery and cardiopulmonary artery bypass grafting. A clear 2- to 3-fold increase in protein carbonyls in serum samples taken from human venous coronary sinus could be detected in the reperfusion period of the heart. We compared these data with markers of oxidative stress previously used, such as the glutathione status and the lipid peroxidation product malondialdehyde (MDA). Strong correlations of the protein carbonyl formation with MDA (r2 = 0.86) and oxidized glutathione (r2 = 0.81) were found in the early reperfusion stage. Increased levels of oxidized glutathione and MDA were detected only in the early reperfusion period. In contrast, the serum protein carbonyl content remained elevated for several hours, indicating a considerably slower serum clearance of oxidized proteins compared with that of lipid peroxidation products and the normalization of the glutathione status. We therefore concluded that the measurement of serum carbonyls by this ELISA technique is suitable to detect oxidative stress in serum samples of patients. The relative stability of the parameter makes the protein carbonyl detection even more valuable for clinical purposes.     

Breath alkanes as a marker of oxidative stress in different clinical conditions

We assessed oxidative stress in three different clinical conditions: smoking, human immunodeficiency virus (HIV) infection, and inflammatory bowel disease, using breath alkane output and other lipid peroxidation parameters such as plasma lipid peroxides (LPO) and malondialdehyde (MDA). Antioxidant micronutrients such as selenium, vitamin E, C, beta-carotene and carotenoids were also measured. Lipid peroxidation was significantly higher and antioxidant vitamins significantly lower in smokers compared to nonsmokers. Beta-carotene or vitamin E supplementation significantly reduced lipid peroxidation in that population. However, vitamin C supplementation had no effect. In HIV-infected subjects, lipid peroxidation parameters were also elevated and antioxidant vitamins reduced compared to seronegative controls. Vitamin E and C supplementation resulted in a significant decrease in lipid peroxidation with a trend toward a reduction in viral load. In patients with inflammatory bowel disease, breath alkane output was also significantly elevated when compared to healthy controls. A trial with vitamin E and C is underway. In conclusion, breath alkane output, plasma LPO and MDA are elevated in certain clinical conditions such as smoking, HIV infection, and inflammatory bowel disease. This is associated with lower levels of antioxidant micronutrients. Supplementation with antioxidant vitamins significantly reduced these lipid peroxidation parameters. The results suggest that these measures are good markers for lipid peroxidation.     

Comparison of DNA adduct levels associated with oxidative stress in human pancreas

DNA adducts associated with oxidative stress are believed to involve the formation of endogenous reactive species generated by oxidative damage and lipid peroxidation. Although these adducts have been reported in several human tissues by different laboratories, a comparison of the levels of these adducts in the same tissue samples has not been carried out. In this study, we isolated DNA from the pancreas of 15 smokers and 15 non-smokers, and measured the levels of 1,N⁶-etheno(2′-deoxy)guanosine (edA), 3,N⁴-etheno(2′-deoxy)cytidine (edC), 8-oxo-2′-deoxyguanosine (8-oxo-dG), and pyrimido[1,2-]purin-10(3H)-one (m₁G). Using the same DNA, the glutathione S-transferase (GST) M1, GSTT1, and NAD(P)H quinone reductase-1 (NQO₁) genotypes were determined in order to assess the role of their gene products in modulating adduct levels through their involvement in detoxification of lipid peroxidation products and redox cycling, respectively. The highest adduct levels observed were for m₁G, followed by 8-oxo-dG, edA, and edC, but there were no differences in adduct levels between smokers and non-smokers and no correlation with the age, sex or body mass index of the subject. Moreover, there was no correlation in adduct levels between edA and eC, or between edA or edC and m₁G or 8-oxo-dG. However, there was a significant correlation (r=0.76; p<0.01) between the levels of 8-oxo-dG and m₁G in human pancreas DNA. Neither GSTM1 nor NQO₁ genotypes were associated with differences in any of the adduct levels. Although the sample set was limited, the data suggest that endogenous DNA adduct formation in human pancreas is not clearly derived from cigarette smoking or from (NQO₁)-mediated redox cycling. Further, it appears that neither GSTM1 nor GSTT1 appreciably protects against endogenous adduct formation. Together with the lack of correlation between m₁G and edA or edC, these data indicate that the malondialdehyde derived from lipid peroxidation may not contribute significantly to m₁G adduct formation. On the other hand, the apparent correlation between m₁G and 8-oxo-dG and their comparable high levels are consistent with the hypothesis that m₁G is formed primarily by reaction of DNA with a base propenal, which, like 8-oxo-dG, is thought to be derived from hydroxyl radical attack on the DNA.