A review is presented of some of the ways in which electron spin resonance (ESR) spectroscopy may be useful to investigate systems of relevance to the biomedical sciences. Specifically considered are: spin-trapping in biological media; the determination of antioxidant efficiencies; lipid-peroxidation; the use of nitroxides as probes of metabolic activity in cells and as structural probes of cell-membranes; ESR coupled with materials for radiation-dosimetry; food-and drug-irradiation; studies of enzyme systems and of cyclodextrins; diagnosis of cancer and rheumatoid arthritis, measurement of oxidative stress in synovial tissue in preparation for joint replacement; determination of oxidative species during kidney dialysis; measurement of biological oxygen concentrations (oximetry); trapping in living cells of the endothelium-derived relaxing factor nitric oxide (NO); measurement of hydrogen peroxide; determination of drugs of abuse (opiates); ESR measurements of whole blood and as a means to determine the age of bloodstains for forensic analysis are surveyed, and also a determination of the aqueous volume of human sperm cells is described, among other topics. Keywords: electron spin resonance, antioxidant efficiencies, lipid-peroxidation, structural probe of cell-membranes, diagnosis of cancer and rheumatoid arthritis, biological fluids 1. Introduction Electron spin resonance (ESR)--also known as electron paramagnetic resonance (EPR) is often viewed as the Cinderella sister of nuclear magnetic resonance (NMR). This is partly because both the application and availability of NMR spectrometers are more apparent, and that not uncommonly ESR receives only passing mention in university courses, unless there is a specialist in the subject on the staff. In principle, NMR spectra may be recorded from dozens of different nuclei, whereas obtaining an ESR spectrum requires some of the sample molecules to contain one or more unpaired electrons, which might appear to be an oddity. Notwithstanding, here lies the beauty of ESR, since the method is completely specific for unpaired electrons, which are frequently formed in materials that have encountered one or more of a range of important energetic conditions for which a signature is supplied in the form of the consequent ESR spectrum. The unpaired electron bearing sites are usually termed "damage centres", "defects" or "trapped electrons" by physicists, geologists, archeologists and environmental scientists, but are normally referred to by chemists and biologists as "free radicals", whose detailed molecular structures may be revealed from the spectral parameters of hyperfine splitting, where it is observed, and of g-factor. Paramagnetic transition-metal cations, e.g. [Fe.sup.3+], [Mn.sup.2+], [Cu.sup.2+], are commonly detected in environmental samples and in biological tissues using ESR. Paramagnetic materials, including metal cations and synthesised complexes or stable organic radicals (usually nitroxides), may be deliberately added to samples as probes of local molecular environments such as cell-membranes, and to determine their dynamic properties. Along with other stable "organic" radicals (e.g. carbon chars and lithium phthalocyanines), nitroxides may be used to measure oxygen tensions in whole tissues and in simpler cellular systems. Metabolic activities in such biological media may also be determined from the reduction kinetics of nitroxides. When it is desired to investigate various reacting systems for the intermediacy of free radicals, "spin-traps" are often added. These are frequently of a structural type designed to "trap" reactive radicals by addition to them, so forming nitroxides in situ, which rise to detectable concentrations in consequence of their relative stability. Clearly, there are many and varied important applications for ESR, and most importantly so in areas of the biological and environmental sciences. 2. …

Electron spin resonance. Part one: a diagnostic method in the biomedical sciences.

Epidemiologists have observed a positive association between human morbidity and mortality and the atmospheric concentrations of fine particulate matter (PM), but the mechanisms underlying the toxic effects of PM have not been elucidated. Various components of ambient PM have been implicated in toxicity (including ultrafine particles, transition metals, organics and oxidants). Our research focused on hydrogen peroxide (H2O2). We speculated that fine PM transports H2O2 into the lower lung, leading to tissue injury and to accumulation and activation of macrophages in these regions. The macrophages release cytotoxic mediators and proinflammatory cytokines that contribute to the pathogenesis of tissue injury. To test this hypothesis, we conducted studies to determine (1) whether tissue injury induced by aerosols is mediated by cytotoxic H2O2 carried into the lower lung by fine particles and (2) whether exposure of rats to fine PM leads to accumulation of activated macrophages in the lung. For our studies, systems were designed to generate model atmospheric fine PM and atmospheric peroxides consisting of an ammonium sulfate [(NH4)2SO4] aerosol (mass median diameter, 0.46 +/- 0.14 microm) and H2O2. We also constructed a 6-port nose-only exposure chamber. Female Sprague Dawley rats were exposed for 2 hours to aerosols consisting of (NH4)2SO4 (430 microg/m3), (NH4)2SO4 + 10, 20 or 100 ppb H2O2, vapor-phase H2O2 (10, 20 or 100 ppb), or particle-free air. Studies using oxygen-18 (18O)-labeled H2O2 were conducted to validate the transport of H2O2 into the lower lung with (NH4)2SO4. Rats were killed immediately (0 hours) or 24 hours after exposure. Compared with control animals, inhalation of (NH4)2SO4 and H2O2, alone or in combination, had no major effect on cell number or viability, protein content, or lactate dehydrogenase (LDH) levels in bronchoalveolar lavage (BAL) fluid collected either immediately or 24 hours after exposure. However, electron microscopy revealed that a larger number of neutrophils in pulmonary capillaries adhered to the vascular endothelium, especially in lungs of rats exposed to (NH4)2SO4 + H2O2. Inhalation of (NH4)2SO4 + H2O2 was also found to be associated with altered macrophage functional activity. Thus, exposing rats to (NH4)2SO4 + 20 ppb H2O2 or 20 ppb H2O2 alone caused a level of tumor necrosis factor alpha (TNF-alpha) production by lung macrophages that was higher than in controls. This higher level was observed immediately after exposure and persisted for at least 24 hours. Greater TNF-alpha production was also detected 24 hours after exposure to (NH4)2SO4 + 10 ppb H2O2. Immediately after rats inhaled (NH4)2SO4 + 10 ppb H2O2 or 20 ppb H2O2 alone, we also observed a transiently higher production of superoxide anion (O2-) by alveolar macrophages. Macrophages isolated 24 hours after exposure to 20 ppb H2O2 also produced larger quantities of superoxide anion. In contrast, immediately after exposure, macrophages from rats exposed to (NH4)2SO4 + 10 ppb H2O2 or to 20 ppb H2O2 alone generated less nitric oxide (NO). Reduced nitric oxide production was also observed 24 hours after exposure to (NH4)2SO4 + 10 ppb H2O2 or to 10 or 20 ppb H2O2 alone. Reduced nitric oxide production may have been due to superoxide anion-driven formation of peroxynitrite (ONOO-) anions. In this regard, nitrotyrosine, an in vivo marker of peroxynitrite, was detected in lung tissue immediately after rats were exposed to (NH4)2SO4 + H2O2 or to H2O2 alone (10 or 20 ppb). We also found that alveolar macrophages from rats exposed to (NH4)2SO4 + H2O2 showed a greater expression of the antioxidant enzyme heme oxygenase-1 (HO-1) when stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Similar results were observed after exposure of rats to an organic peroxide aerosol (cumene hydroperoxide). Taken together, the results of our studies demonstrate that biological effects of inhaled H2O2 are augmented by fine PM. Moreover, tissue injury induced by (NH4)2SO4 + H2O2 may be related to altered production of cytotoxic mediators by alveolar macrophages. Determining the relevance of these toxicologic results to human health will be important in future studies for evaluating the risk of exposure.

Peroxides and macrophages in the toxicity of fine particulate matter in rats.

Cumene hydroperoxide debilitates macrophage physiology by inducing oxidative stress: possible protection by α-tocopherol.

While inhalation of ozone is known to cause a variety of health effects, the reactions at a molecular level that lead to these effects are not well understood. One potential path is the reaction of ozone with the unsaturated fatty acid components of pulmonary surfactant at the air-water interface in the lung to form secondary ozonides. These have been proposed to decompose to free radicals, which can then initiate the well-known inflammatory response. We report here the first kinetic studies of the thermal decomposition of the cis and trans secondary ozonides of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a phospholipid found in significant quantities in lung surfactant. The ozonides were synthesized by reaction of O3 with POPC adsorbed on a glass surface, and their thermal decomposition kinetics were followed using high-performance liquid chromatography (HPLC) over the temperature range from 50°C to 106°C in either methanol or 1,1,1,2-tetrachloroethane. The Arrhenius parameters for the thermal decomposition in methanol are A = 10(6.7±0.3) s-1 and E(a) = 19.6 ± 0.6 kcal mol-1 for the cis ozonide, and A = 10(8.7±0.6) s-1 and E(a) = 19.8 ± 1.0 kcal mol-1 for the trans ozonide. In 1,1,1,2-tetrachloroethane, the parameters are A = 10(8.3±2.1) s-1 and E(a) = 18.4 ± 3.4 kcal mol-1 for the cis ozonide, and A = 10(9.3±3.2) s-1 and E(a) = 20.2 ± 5.2 kcal mol-1 for the trans ozonide (all errors cited are ±2σ). Within experimental error, there is no difference in the kinetics of decomposition in the two solvents. However, both the activation energy and the preexponential factor for the decomposition of the phospholipid ozonides are significantly lower than those for decomposition of the long-chain alkene ozonide 1-octene ozonide, determined to be E(a) = 26.7 ± 3.2 kcal mol-1 and A = 10(12.7±1.9) s-1. The latter reaction has been proposed to be initiated by scission of the O-O bond, followed by decomposition of the peroxy biradical to generate free radicals. The kinetics for the decomposition of the POPC ozonides in solution are similar to those of simple alkene ozonides in the gas phase, where a concerted mechanism involving simultaneous intramolecular hydrogen transfer and O-O bond cleavage has been proposed. The only high-molecular-weight major product of the POPC ozonide decomposition identified using liquid secondary ion mass spectrometry (LSIMS) was the lipid acid 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine, which was observed as a product in both solvents. The mechanism and implications for the toxicology of inhaled ozone are discussed.

https://escholarship.org/content/qt5nr1k5br/qt5nr1k5br.pdf?t=obni5u

H.N.A. Grooten

Papers

Comparative study on the toxicity of methyl linoleate-9,10-ozonide and cumene hydroperoxide to alveolar macrophages.