Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.

Plasma and urinary levels of malondialdehyde-like products (MDA) and isoprostanes were identified as markers of in vivo lipid peroxidation in an animal model of CCl4 poisoning. We sought to determine the extent to which the formation of these oxidation products is influenced by inhibition of the cyclooxygenase enzymes which catalytically generate proinflammatory lipid peroxidation products known as prostaglandins and thromboxane. In the present studies, after induction of oxidant stress in rats with CCl4, lipid peroxidation products measured in plasma and urine demonstrate that isoprostanes and MDA can be partially inhibited by cyclooxygenase inhibitors, albeit to different extents. The lowering of isoprostane and MDA formation, however, may not to due primarily to the diminution of catalytic generation of isoprostanes or MDA by the cyclooxygenases but, rather, may be the result of the suppression of nonenzymatic lipid peroxidation. This is suggested since 8,12-iso-iPF2α−VI is also reduced by indomethacin, yet, unlike other isoprostanes and MDA, it is not generated catalytically by the cyclooxygenase. Thus, although the two cyclooxygenase inhibitors we tested have statistically significant effects on the measurements of both isoprostanes and MDA in this study, the results provide evidence that these lipid-degradation products primarily constitute markers of oxidative stress.

Gamma-tocopherol (gammaT) complements alpha-tocopherol (alphaT) by trapping reactive nitrogen oxides to form a stable adduct, 5-nitro-gammaT [Christen et al., PNAS 94:3217-3222; 1997]. This observation led to the current investigation in which we studied the effects of gammaT supplementation on plasma and tissue vitamin C, vitamin E, and protein nitration before and after zymosan-induced acute peritonitis. Male Fischer 344 rats were fed for 4 weeks with either a normal chow diet with basal 32 mg alphaT/kg, or the same diet supplemented with approximately 90 mg d-gammaT/kg. Supplementation resulted in significantly higher levels of gammaT in plasma, liver, and kidney of control animals without affecting alphaT, total alphaT+gammaT or vitamin C. Intraperitoneal injection of zymosan caused a marked increase in 3-nitrotyrosine and a profound decline in vitamin C in all tissues examined. Supplementation with gammaT significantly inhibited protein nitration and ascorbate oxidation in the kidney, as indicated by the 29% and 56% reduction of kidney 3-nitrotyrosine and dehydroascorbate, respectively. Supplementation significantly attenuated inflammation-induced loss of vitamin C in the plasma (38%) and kidney (20%). Zymosan-treated animals had significantly higher plasma and tissue gammaT than nontreated pair-fed controls, and the elevation of gammaT was strongly accentuated by the supplementation. In contrast, alphaT did not significantly change in response to zymosan treatment. In untreated control animals, gammaT supplementation lowered basal levels of 3-nitrotyrosine in the kidney and buffered the starvation-induced changes in vitamin C in all tissues examined. Our study provides the first in vivo evidence that in rats with high basal amounts of alphaT, a moderate gammaT supplementation attenuates inflammation-mediated damage, and spares vitamin C during starvation-induced stress without affecting alphaT.

γ-Tocopherol is the major form of vitamin E in many plant seeds and in the US diet, but has drawn little attention compared with α-tocopherol, the predominant form of vitamin E in tissues and the primary form in supplements. However, recent studies indicate that γ-tocopherol may be important to human health and that it possesses unique features that distinguish it from α-tocopherol. γ-Tocopherol appears to be a more effective trap for lipophilic electrophiles than is α-tocopherol. γ-Tocopherol is well absorbed and accumulates to a significant degree in some human tissues; it is metabolized, however, largely to 2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxychroman (γ-CEHC), which is mainly excreted in the urine. γ-CEHC, but not the corresponding metabolite derived from α-tocopherol, has natriuretic activity that may be of physiologic importance. Both γ-tocopherol and γ-CEHC, but not α-tocopherol, inhibit cyclooxygenase activity and, thus, possess antiinflammatory properties. Some human and animal studies indicate that plasma concentrations of γ-tocopherol are inversely associated with the incidence of cardiovascular disease and prostate cancer. These distinguishing features of γ-tocopherol and its metabolite suggest that γ-tocopherol may contribute significantly to human health in ways not recognized previously. This possibility should be further evaluated, especially considering that high doses of α-tocopherol deplete plasma and tissue γ-tocopherol, in contrast with supplementation with γ-tocopherol, which increases both. We review current information on the bioavailability, metabolism, chemistry, and nonantioxidant activities of γ-tocopherol and epidemiologic data concerning the relation between γ-tocopherol and cardiovascular disease and cancer.

Zymosan-induced peritonitis is associated with an increased production of reactive nitrogen oxides that may contribute to the often-observed failure of multiple organ systems in this model of acute inflammation. Quantitative biochemical evidence is provided for a marked 13-fold increase in protein-bound 3-nitrotyrosine (NTyr), a biomarker of reactive nitrogen oxides, in liver tissue of zymosan-treated rats. In order to investigate the localization of NTyr in this affected tissue, a monoclonal antibody, designated 39B6, was raised against 3-(4-hydroxy-3-nitrophenylacetamido) propionic acid-bovine serum albumin conjugate and its performance characterized. 39B6 was judged by competition ELISA to be approximately 2 orders of magnitude more sensitive than a commercial anti-NTyr monoclonal antibody. Binding characteristics of 39B6 were similar, but not identical, to that of a commercial affinity-purified polyclonal antibody in ELISA and immunohistochemical analyses. Western blot experiments revealed high specificity of 39B6 against NTyr and increased immunoreactivity of specific proteins from liver tissue homogenates of zymosan-treated rats. Immunohistochemical analysis of liver sections indicated a marked zymosan-induced increase in immunofluorescent staining, which was particularly intense in or adjacent to nonparenchymal cells, but not in the parenchymal cells of this tissue. Quantitative analysis of fractions enriched in these cell populations corroborated the immunofluorescent data, although the relative amounts detected in response to zymosan treatment was greatly reduced compared to whole liver tissue. These results demonstrate the high specificity of the newly developed antibody and its usefulness in Western blot and immunohistochemical analysis for NTyr, confirm the presence of NTyr by complementary methods, and suggest the possible involvement of reactive nitrogen oxides in hepatic vascular dysfunction.

Cystic fibrosis (CF) is associated with chronic pulmonary inflammation and progressive lung dysfunction, possibly associated with the formation of neutrophil myeloperoxidase (MPO)-derived oxidants. Expectorated sputum specimens from adult CF patients were analyzed for MPO characteristic protein modifications and found to contain large amounts of active MPO as well as high levels of protein-associated 3-chlorotyrosine and 3,3'-dityrosine, products that result from MPO activity, compared with expectorated sputum from non-CF subjects. Sputum levels of nitrite (NO(2)(-)) and nitrate (NO(3)(-)), indicating local production of nitric oxide (NO. ), were not elevated but in fact were slightly reduced in CF. However, there was a slight increase in protein-associated 3-nitrotyrosine in CF sputum compared with controls, reflecting the formation of reactive nitrogen intermediates, possibly through MPO-catalyzed oxidation of NO(2)(-). CF sputum MPO was found to contribute to oxidant-mediated cytotoxicity toward cultured tracheobronchial epithelial cells; however, peroxidase-dependent protein oxidation occurred primarily within sputum proteins, suggesting scavenging of MPO-derived oxidants by CF mucus and perhaps formation of secondary cytotoxic products within CF sputum. Our findings demonstrate the formation of MPO-derived oxidizing and possibly nitrating species within the respiratory tract of subjects with CF, which collectively may contribute to bronchial injury and respiratory failure in CF.

Ataxia-telangiectasia (A-T) is characterized by a markedly increased sensitivity to ionizing radiation, increased incidence of cancer, and neurodegeneration, especially of the cerebellar Purkinje cells. Ionizing radiation oxidizes macromolecules and causes tissue damage through the generation of reactive oxygen species (ROS). We therefore hypothesized that A-T is due to oxidative damage resulting from loss of function of the A-T gene product. To assess this hypothesis, we employed an animal model of A-T, the mouse with a disrupted Atm gene. We show that organs which develop pathologic changes in the Atm-deficient mice are targets of oxidative damage, and that cerebellar Purkinje cells are particularly affected. These observations provide a mechanistic basis for the A-T phenotype and lay a rational foundation for therapeutic intervention.

HPLC with electrochemical detection of the N-acetylated, dithionite-reduced derivative of NTyr provides a highly sensitive and selective means of measuring this nitrated residue in biological samples. The detection of protein-bound NTyr at baseline levels of approximately < or = 1 mumol per mole Tyr indicates that in plasma or total cellular extracts, endogenous nitration of tyrosine residues is low. This baseline level of NTyr and the marked increases that are observed during inflammatory conditions opens up the opportunity to observe more subtle changes in tyrosine nitration, thus broadening the range of studies that can be performed using this biomarker. This analytical approach may allow one to estimate protein nitration in an animal or individual exposed to elevated levels of peroxynitrite or other reactive nitrogen oxides, and it may assist in the evaluation of factors that contribute to this potentially important amino acid modification. Furthermore, this assay may allow one to assess the potential benefits of interventions that may limit nitration reactions in vivo.

Since its discovery as a biologic messenger molecule just over a decade ago, nitric oxide (NO · ) has become well recognized for its participation in diverse biologic processes in nearly all aspects of life, including vasodilation, bronchodilation, neurotransmission, inhibition of phagocyte and platelet aggregation, and antimicrobial activity (1–3). Excessive production of NO · during inflammatory–immune processes of the respiratory tract is thought to provide a host defense mechanism, although this comes with a price, since high levels of NO · can also cause respiratory tract injury and thus contribute to the pathobiology of respiratory tract disease. These detrimental effects of NO · are generally assumed to be related to the formation of more reactive nitrogen intermediates via interactions of NO · with partially reduced oxygen species, a common hallmark of inflammatory processes. Conversely, NO · has in some cases been shown to also attenuate oxidant-induced lung injury, and NO · inhalation has been proposed as a therapeutic strategy in the management of pulmonary hypertension and in some forms of adult respiratory distress syndrome (ARDS). This dual property of NO · has been the subject of intense recent investigation, which has uncovered multifaceted biochemical pathways of NO · that are highly dependent on dose and on local redox status. NO · -derived reactive nitrogen intermediates can induce a number of covalent modifications in various biomolecules, such as nitrosoand nitroadducts, that result in functional and/or structural changes. One such modification yields 3-nitrotyrosine, and detection of this adduct in proteins is now commonly used as a diagnostic tool to identify involvement of NO · -derived oxidants in many disease states (4–6). Furthermore, a number of in vitro studies have established changes in enzyme activity upon nitration of critical tyrosine residues, which has raised suggestions that protein nitration in vivo may be causally linked to inflammation-related forms of lung injury. In this Pulmonary Perspective we will briefly summarize the involvement of NO · in the pathophysiology of inflammatory diseases of the respiratory tract, and will address characteristic diagnostic modifications in proteins or in other biomolecules, with special emphasis on 3-nitrotyrosine and related modifications in other aromatic substrates. We will examine the scope of bioreactive mechanisms known to contribute to aromatic nitration during inflammatory–immune processes, and will critically evaluate analytical procedures that have been developed and used to detect such modifications. We will also discuss the potential pathophysiologic consequences of tyrosine nitration and related modifications.

Abstract This chapter discusses the possibility that nicotine, and/or some of the minor tobacco alkaloids, may be biotransformed to chemically reactive intermediates that, following chronic exposure, could give rise to health hazards, including cancer. It will be useful to define the metabolic profile of (S)-nicotine in order to identify possible chemical links to the biological properties of this important component. This chapter examines the pathways responsible for these conversions and the metabolic fate of the cyclic tertiary amine (S)-nicotine (1) and its pyrrolic analognicotyrine (2) (Fig. 6-1), with particular emphasis on the possible formation of chemically reactive metabolites that may contribute to the adverse health outcomes associated with chronic tobacco use.

Oxidative DNA damage is important in aging and the degenerative diseases of aging such as cancer, Estimates commonly rely on measurements of 8-oxo-2'-deoxyguanosine (oxo(8)dG), an adduct that occurs in DNA and is also excreted in urine after DNA repair, Here we examine difficulties inherent in the analysis of oxo(8)dG, identify sources of artifacts, and provide solutions to some of the common methodological problems, A frequent criticism has been that phenol in DNA extraction solutions artificially increases the measured level of oxo(8)dG, We found that phenol extraction of DNA contributes a real but minor increase in the level of oxo(8)dG when compared, under equivalent conditions, with a successful nonphenol method, A more significant reduction in the baseline level was achieved with a modification of the recently introduced chaotropic NaI method, reducing our estimate of the level of steady-state oxidative adducts by an order of magnitude to 24,000 adducts per cell in young rats and 66,000 adducts per cell in old rats, Of several alternative methods tested, the use of this chaotropic technique of DNA isolation by using NaI produced the lowest and least variable oxo(8)dG values. In further studies we show that human urinary 8-oxo-guanine (oxo(8)Gua) excretion is not affected by the administration of allopurinol, suggesting that, unlike some methylated adducts, oxo(8)Gua is not derived enzymatically from xanthine oxidase, Lastly, we discuss remaining uncertainties inherent both in steady-state oxo(8)dG measurements and in estimates of endogenous oxidation ("hit rates") based on urinary excretion of oxo(8)dG and oxo(8)Gua.

The identification of 15N-labeled 3-nitrotyrosine (NTyr) by gas chromatography/mass spectroscopy in protein hydrolyzates from activated RAW 264.7 macrophages incubated with 15N-L-arginine confirms that nitric oxide synthase (NOS) is involved in the nitration of protein-bound tyrosine (Tyr). An assay is presented for NTyr that employs HPLC with tandem electrochemical and UV detection. The assay involves enzymatic hydrolysis of protein, acetylation, solvent extraction, O-deacetylation, and dithionite reduction to produce an analyte containing N-acetyl-3-aminotyrosine, an electrochemically active derivative of NTyr. We estimate the level of protein-bound NTyr in normal rat plasma to be approximately 0-1 residues per 10(6) Tyr with a detection limit of 0.5 per 10(7) Tyr when > 100 nmol of Tyr is analyzed and when precautions are taken to limit nitration artifacts. Zymosan-treated RAW 264.7 cells were shown to have an approximately 6-fold higher level of protein-bound NTyr compared with control cells and cells treated with N(G)-monomethyl-L-arginine, an inhibitor of NOS. Intraperitoneal injection of F344 rats with zymosan led to a marked elevation in protein-bound NTyr to approximately 13 residues per 10(6) Tyr, an approximately 40-fold elevation compared with plasma protein of untreated rats; cotreatment with N(G)-monomethyl-L-arginine inhibited the formation of NTyr in plasma protein from blood and peritoneal exudate by 69% and 53%, respectively. This assay offers a highly sensitive and quantitative approach for investigating the role of reactive byproducts of nitric oxide in the many pathological conditions and disease states associated with NO(X) exposure such as inflammation and smoking.

Peroxynitrite, a powerful mutagenic oxidant and nitrating species, is formed by the near diffusion-limited reaction of ·NO and O 2⨪ during activation of phagocytes. Chronic inflammation induced by phagocytes is a major contributor to cancer and other degenerative diseases. We examined how γ-tocopherol (γT), the principal form of vitamin E in the United States diet, and α-tocopherol (αT), the major form in supplements, protect against peroxynitrite-induced lipid oxidation. Lipid hydroperoxide formation in liposomes (but not isolated low-density lipoprotein) exposed to peroxynitrite or the ·NO and O 2⨪ generator SIN-1 (3-morpholinosydnonimine) was inhibited more effectively by γT than αT. More importantly, nitration of γT at the nucleophilic 5-position, which proceeded in both liposomes and human low density lipoprotein at yields of ≈50% and ≈75%, respectively, was not affected by the presence of αT. These results suggest that despite αT’s action as an antioxidant γT is required to effectively remove the peroxynitrite-derived nitrating species. We postulate that γT acts in vivo as a trap for membrane-soluble electrophilic nitrogen oxides and other electrophilic mutagens, forming stable carbon-centered adducts through the nucleophilic 5-position, which is blocked in αT. Because large doses of dietary αT displace γT in plasma and other tissues, the current wisdom of vitamin E supplementation with primarily αT should be reconsidered.