Ronald P. Mason - US grants

animal tissue; spectrometry; biomedical resource; biomedical equipment development;

Oxidative damage to tissues by hydrogen peroxide has become a recurring theme as a mechanism for the induction of a variety of medical conditions including myocardial ischemia, cancer, inflammation, and aging. Proteins are a target of such damage. Most heme-containing proteins have peroxidase activity that can cause oxidative damage to a variety of biological molecules, including itself and membrane lipids. We are now in the process of investigating a series of protein-derived radicals formed by myoglobin, hemoglobin and cytochrome c oxidase that utilize our new immunological-spin trapping techniques. Our laboratory has produced polyclonal antibodies that bind specifically to the spin trap/protein adduct of 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The antibodies were produced in rabbits immunized against the hapten, 5,5-dimethyl-2-octanoic acid-1-pyrroline N-oxide (OA-DMPO). OA-DMPO is a structural analog of DMPO with an additional carbon chain terminating in a carboxyl-group for amide linking. Antigens were synthesized by reacting OA-DMPO with chicken egg albumin via the carbodiimide method for amidation. Titer of the rabbit serum was determined using ELISA techniques. The polyclonal antibodies screened positive for protein-DMPO nitrone adducts produced on BSA via Fenton chemistry and adducts produced on metmyoglobin as a result of self-peroxidation by hydrogen peroxide.

Previous reports have examined several proposed markers of oxidative stress in a model system of CCl4 administered to rats. Our work on the CCl4 model has identified a lower GSH/GSSG ratio as an early marker of oxidative stress, and lipid peroxidation products of plasma and urine MDA and F2-isoprostanes were reported as the most reliable, sensitive and accurate markers for measurement of oxidative stress since they were both increased in a time-and dose-dependent pattern. In the present study, rodent inhalation exposure to ozone was chosen as the second model of oxidative stress to determine whether exposure to ozone causes oxidative lung damage that can be quantitated by the identification and measurement of the presence of various products of oxidation in blood, plasma or urine. Although the molecular mechanisms of ozone-induced lung cell injury are not fully understood, one hypothesis is that ozone reacts with cell membrane lipids, inducing lipid peroxidation, aldehyde production and generation of lipid ozonation products that are considered to be involved in the early stage of inflammatory response. The first objective of this study was to determine whether acutely exposing rats to ozone would result in the loss of antioxidants from plasma and bronchoalveolar lavage fluid (BALF). Additional goals were to compare analyses of the same antioxidant concentration between different laboratories, to investigate which methods have the sensitivity to detect decreased levels of antioxidants, and to identify a reliable measure of oxidative stress in ozone-exposed rats. Male Fisher rats were exposed to either 2.0 or 5.0 ppm ozone inhalation for 2 h. Blood plasma and BALF samples were collected 2, 7, and 16 h after the exposure. It was found that ascorbic acid in plasma collected from rats after the higher dose of ozone was lower at 2 h, but not later. BALF concentrations of ascorbic acid were decreased at both 2 and 7 h post-exposure. Tocopherols, 5-nitro--tocopherol, tocol, glutathione (GSH/GSSG), and cysteine (Cys/CySS) were not decreased, regardless of the dose or post-exposure time point used for sample collection. Uric acid was significantly increased by the low dose at 2 h and the high dose at the 7 h point, probably because of the accumulation of blood plasma in the lung from ozone-increased alveolar capillary permeability. We conclude that measurements of antioxidants in plasma are not sensitive biomarkers for oxidative damage induced by ozone and are not a useful choice for the assessment of oxidative damage by ozone in vivo. The second objective of the ozone exposure study was to determine whether oxidation products of lipids, proteins and DNA could be identified as markers of oxidative stress. The time- and dose-dependent effects of ozone exposure on rat plasma lipid hydroperoxides, malondialdehyde, F2-isoprostanes, protein carbonyls, methionine oxidation, tyrosine and phenylalanine oxidation products, as well as urinary malondialdehyde and F2-isoprostanes, were investigated again with various techniques. The criterion used to recognize a marker in the model of ozone exposure was whether or not a significant effect could be identified and measured in a biological fluid seen at both doses at more than one time point. No statistically significant differences between the experimental and control groups at either ozone dose or time point studied could be identified in this study. Tissue samples were not included. Despite all the work accomplished in the BOSS study of ozone, no available product of oxidation in biological fluid has yet met the required criterion of being a biomarker. The current negative findings as a consequence of ozone exposure are of great importance, because they document that in complex systems, as in the present in vivo experiment, the assays used may not provide meaningful data of ozone oxidation, especially in human studies. The major conclusionof these studies is that the detection of oxidative stress markers in animal models is exceedingly difficult even in models near the LD50 level. These publications, which are highly cited, have undoubtedly saved other investigators both time and money, especially in epidemiological studies. The biomarker 8-iso-prostaglandin F2 (8-iso-PGF2) is regarded as the gold standard for detection of excessive chemical lipid peroxidation in humans. We found that increase in 8-iso-PGF2 do not necessarily reflect in oxidative stress; therefore, past studies using 8-iso-PGF2 as a marker of oxidative stress may have been misinterpreted. The 8-iso-PGF2/PGF2 ratio can be used to distinguish biomarker synthesis pathways and thus confirm the potential change in oxidative stress in the myriad of disease and chemical exposures known to induce 8-iso-PGF2. We investigated the formation of 8-iso-PGF2 in rats exposed to carbon tetrachloride(CCl4) or lipopolysaccharide (LPS) using the 8-iso-PGF2/PGF2 ratio to quantitatively determine the source(s)of 8-iso-PGF2. Upon exposure to a 120mg/kg dose of CCl4, the contribution of CLP accounted for only 55.6+/-19.4% of measured 8-iso-PGF2, whereas in the 1200mg/kg dose, CLP was the predominant source of 8-iso-PGF2 (86.6+/-8.0% of total).In contrast to CCl4, exposure to 0.5mg/kg LPS was characterized by a significant increase in both the contribution of PGHS(59.5+/-7.0) and CLP(40.5+/-14.0%). In conclusion, significant generation of 8-iso-PGF2 occurs through enzymatic as well as chemical lipid peroxidation. The distribution of the contribution is dependent on the exposure agent as well as the dose. The8-iso-PGF2/PGF2 ratio accurately determines the source of 8-iso-PGF2 and provides an absolute measure of oxidative stress in vivo. The notion that oxidative stress plays a role in virtually every human disease and environmental exposure has become ingrained in everyday knowledge. However, mounting evidence regarding the lack of specificity of biomarkers traditionally used as indicators of oxidative stress in human disease and exposures now necessitates re-evaluation. To prioritize these re-evaluations, published literature was comprehensively analyzed in a meta analysis to quantitatively classify the levels of systemic oxidative damage across human disease and in response to environmental exposures. In this meta-analysis, the F2-isoprostane, 8-iso-PGF2, was specifically chosen as the representative marker of oxidative damage. To combine published values across measurement methods and specimens, the standardized mean differences (Hedges g) in 8-iso-PGF2 levels between affected and control populations were calculated. The meta-analysis resulted in a classification of oxidative damage levels as measured by 8-iso-PGF2 across 50 human health outcomes and exposures from 242 distinct publications. Relatively small increases in 8-iso-PGF2 levels (g<0.8) were found in the following conditions: hypertension (g=0.4), metabolic syndrome (g=0.5), asthma (g=0.4), and tobacco smoking (g=0.7). In contrast, large increases in 8-iso-PGF2 levels were observed in pathologies of the kidney, e.g., chronic renal insufficiency (g=1.9), obstructive sleep apnoea (g=1.1), and pre-eclampsia (g=1.1), as well as respiratory tract disorders, e.g., cystic fibrosis (g=2.3). In conclusion, we have established a quantitative classification for the level of 8-iso-PGF2 generation in different human pathologies and exposures based on a comprehensive meta-analysis of published data. This analysis provides knowledge on the true involvement of oxidative damage across human health outcomes as well as utilizes past research to prioritize those conditions requiring further scrutiny on the mechanisms of biomarker generation.

Spin Trapping has been the most successful method for the detection of highly reactive free radical molecules in vivo. This technique involves the direct detection of primary free radicals that cannot be directly observed by conventional ESR due to low steady-state concentrations or to very short relaxation times, which lead to very broad lines. All the reported in vivo spin-trapping investigations have used the nitrone spin traps (PBN, POBN, DMPO). Although the major difficulty of the spin-trapping technique in vivo is the mere detection of a radical adduct, other factors such as absorption, distribution, metabolism and excretion must be considered when spin traps are administered in vivo. In addition, artifacts and ambiguities in the assignment of radical adduct structure must be taken into serious consideration. The group has pioneered the application of the ESR spin-trapping technique to biochemical, pharmacological and toxicological problems by using two major approaches for in vivo spin-trapping and ex vivo ESR detection of free radicals: lipid extraction of radical adducts generated in tissues and detection of radical adducts in biological fluids. [unreadable] i. Metal-mediated Free Radical Production: ESR spin-trapping investigations of acute cadmium- and/or diesel exhaust particle poisoning have shown the in vivo formation of either the hydroxyl radical in the liver or lipid-derived radicals in the lungs. [unreadable] ii. Characterization of Endogenous Radical Adducts by HPLC/ESR/MS: The combination of an on-line high performance liquid chromatography (HPLC)/electron spin resonance (ESR) system with mass spectrometric analysis (MS) was used to identify a variety of lipid-derived radicals formed from lipid peroxidation both in vitro and in vivo. For the first time, POBN adducts of linoleic acid carbon-centered pentadienyl radicals were detected and identified. [unreadable] iii. Novel Methanol and Ethylene Glycol Free Radical Metabolites: The investigations of ethanol's free radical metabolism have been extended using knockout mice, chemical analogues such as ethylene glycol and methanol, and their metabolites such as formic acid. The evidence for formate-derived radical metabolites in vivo, during acute sodium formate poisoning is provided by ESR spin trapping of the radicals from the metabolism of formate, which are detected in bile and urine as radical adducts.