Jean T. Corbett

In vivo lipid-derived free radical formation by NADPH oxidase in acute lung injury induced by lipopolysaccharide: a model for ARDS

Intratracheal instillation of lipopolysaccharide (LPS) activates alveolar macrophages and infiltration of neutrophils, causing lung injury/acute respiratory distress syndrome. Free radicals are a special focus as the final causative molecules in the pathogenesis of lung injury caused by LPS. Although in vitro investigation has demonstrated radical generation after exposure of cells to LPS, in vivo evidence is lacking. Using electron spin resonance (ESR) and the spin trap a‐(4‐pyridyl‐1‐oxide)‐N‐tert‐butylnitrone (POBN), we investigated in vivo free radical production by rats treated with intratracheal instillation of LPS. ESR spectroscopy of lipid extract from lungs exposed to LPS for 6 h gave a spectrum consistent with that of a POBN/ carbon‐centered radical adduct (aN=14.94±0.07 G and a:H=2.42±0.06 G) tentatively assigned as a product of lipid peroxidation. To further investigate the mechanism of LPS‐initiated free radical generation, rats were pretreated with the phagocytic toxicant GdCl3, which significantly decreased the production of radical ad‐ducts with a Corresponding decrease in neutrophil infiltration. NADPH oxidase knockout mice completely blocked phagocyte‐mediated, ESR‐detectable radical production in this model of acute lung injury. Rats treated intratracheally with LPS generate lipid‐derived free radicals via activation of NADPH oxidase.—Sato, K., Kadiiska, M. B., Ghio, A. J., Corbett, J., Fann, Y. C., Holland, S. M., Thurman, R. G., Mason, R. P. In vivo lipid‐derived free radical formation by NADPH oxidase in acute lung injury induced by lipopolysaccharide: a model for ARDS. FASEB J. 16, 1713–1720 (2002)

Quantitative characterization of polychlorinated biphenyl mixtures (aroclors® 1248, 1254 and 1269) by gas chromatograpy using capillary columns

Abstract The polychlorinated biphenyl (PCB) compositions of Aroclors 1248, 1254 and 1260 have been determined using gas chromatography. A highly efficient glass capillary coated with teh moderately polar Dexsil 410 served as the primary column. Pairs of isomers not resolved on Dexsil 410 were easily distiguished on short, less efficient capillaries coated with Silar 5c, Apiezon L, or OV-25. The advantages associated with the high selectivity of moderately polar columns outweighed the high column efficiencies associated with non-polar phases, permitting not-very-efficient columns to provide the needed information. Meaningful quantitation was obtained through the use of the hydrogen flame ionization detector. These Aroclors, now characterized in terms of the relative molar percentages of approximately 100 different PCBs. can be used as secondary standards to obtain relative molar responses for electron capture detectors. This will permit valid application of quantitative gas- liquid chromatography to environmental samples.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on lipid profiles in tissue of the Fischer rat.

Female Fischer 344 rats were given single oral doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 10, 50 or 100 microgram/kg, and sacrificed 1, 3, 10, 14 or 21 days later. The fatty livers caused by a sub-lethal dose of TCDD involved a temporary increase in triglyceride and free fatty acid levels, with a persistent decrease in levels of sterol esters. In contrast, the fatty livers resulting from a lethal dose of TCDD involved a large increase in cholesterol esters and free fatty acids, with little change in triglyceride levels. These changes appeared to result in part from damage sustained by lysosomes. TCDD also altered the lipoprotein composition of the serum, the fatty acid composition of various lipid classes in liver and serum, and the ultrastructure of the liver (formation of myeloid bodies). A rapid, dose-dependent effect of TCDD, was the elevation of levels of organic-soluble fluorescent pigment in the heart. This pigment was found to match a previously characterized fraction of lipofuscins in fluorescence spectrum and chromatographic properties. The relatioship of these observations to a possible mechanism of toxicity for TCDD involving radical-induced lipid peroxidation is discussed.

Leptin is key to peroxynitrite-mediated oxidative stress and Kupffer cell activation in experimental non-alcoholic steatohepatitis

BACKGROUND & AIMS Progression from steatosis to steatohepatitic lesions is hypothesized to require a second hit. These lesions have been associated with increased oxidative stress, often ascribed to high levels of leptin and other proinflammatory mediators. Here we have examined the role of leptin in inducing oxidative stress and Kupffer cell activation in CCl4-mediated steatohepatitic lesions of obese mice. METHODS Male C57BL/6 mice fed with a high-fat diet (60%kcal) at 16 weeks were administered CCl₄ to induce steatohepatitic lesions. Approaches included use of immuno-spin trapping for measuring free radical stress, gene-deficient mice for leptin, p47 phox, iNOS and adoptive transfer of leptin primed macrophages in vivo. RESULTS Diet-induced obese (DIO) mice, treated with CCl4 increased serum leptin levels. Oxidative stress was significantly elevated in the DIO mouse liver, but not in ob/ob mice, or in DIO mice treated with leptin antibody. In ob/ob mice, leptin supplementation restored markers of free radical generation. Markers of free radical formation were significantly decreased by the peroxynitrite decomposition catalyst FeTPPS, the iNOS inhibitor 1400W, the NADPH oxidase inhibitor apocynin, or in iNOS or p47 phox-deficient mice. These results correlated with the decreased expression of TNF-alpha and MCP-1. Kupffer cell depletion eliminated oxidative stress and inflammation, whereas in macrophage-depleted mice, the adoptive transfer of leptin-primed macrophages significantly restored inflammation. CONCLUSIONS These results, for the first time, suggest that leptin action in macrophages of the steatotic liver, through induction of iNOS and NADPH oxidase, causes peroxynitrite-mediated oxidative stress thus activating Kupffer cells.

Mono-2-ethylhexyl phthalate, a metabolite of di-(2-ethylhexyl) phthalate, causally linked to testicular atrophy in rats

Acute testicular atrophy results when appropriate dosages of di-(2-ethylhexyl) phthalate (DEHP) or its hydrolysis product mono-2-ethylhexyl phthalate (MEHP) are given to male rats. Events thought to be involved in this pathological effect also occur in cultures of testicular cells in vitro, but require MEHP rather than DEHP. Primary cultures of hepatocytes, Sertoli cells, and Leydig cells were incubated with 14C-labeled MEHP [8 microM] for up to 24 hr. No significant reduction in viability was produced under these conditions. In contrast to the hepatocytes, which extensively metabolized MEHP to a variety of products in 1 hr, the testicular cell cultures were apparently unable to metabolize MEHP (beyond a slight hydrolysis to phthalic acid by Sertoli cells) in 18-24 hr. MEHP was efficiently taken up by hepatocytes, but much less so by testicular cells. These results, combined with related observations from the literature, support the hypothesis that MEHP itself is the metabolite of DEHP responsible for testicular atrophy in rats.

The scopoletin assay for hydrogen peroxide A review and a better method

Scopoletin, 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one, a naturally occurring component in cotton leaf and citrus peel is a fluorescent substrate for peroxidase which has been used by many investigators for the determination of hydrogen peroxide concentration. The technical details of these investigations are application-specific and rather critical, making it difficult to apply the scopoletin assay to alternative systems without extensive modification. Although such factors as interfering substances and optimum conditions have been discussed in many publications, these discussions tend to be application-specific. The present paper attempts to provide a technical review of scopoletin applications, add a few new experimental observations, and discuss general parameters which must be carefully controlled for reliable results.

Application of the thiobarbiturate assay to the measurement of lipid peroxidation products in microsomes

By applying two different thiobarbiturate assay procedures in parallel to aliquots of a microsomal incubation mixture one can simultaneously monitor free malondialdehyde and malondialdehyde plus labile lipid peroxidation products. The levels of malondialdehyde increase continuously during the incubation of microsomes, NADPH and ferrous-ADP complex, while the lipid precursors of MDA stop forming when the system becomes depleted in NADPH. In contrast to systems in which lipids are undergoing autooxidation, NADPH-dependent lipid peroxidation does not appear to generate significant amounts of water-soluble malondialdehyde precursors. As a result, quantitative interpretation of results is straightforward in the microsomal system. In spite of the lack of specificity of the thiobarbiturate coupling reaction, interferences can be easily compensated for by using zero time controls.

PHOTOCHEMICAL REACTIONS AND PHOTOTOXICITY OF STEROLS : NOVEL SELF-PERPETUATING MECHANISM FOR LIPID PHOTOOXIDATION

Abstract— Sterols are important lipid components that may contribute to phototoxicity. We have found that phototoxic response in earthworms is related to sterols extractable with lipophilic solvents. The photochemically active compounds in worm lipids are 5,7,9(11),22‐ergostatetraen‐3bT‐ol (9‐DHE) and 5,7,9(11)‐cholestatrien‐3bT‐ol (9‐DDHC), respectively. Human skin lipids are known to contain 9‐DHE. We have also found 9‐DDHC in human skin, which is reported here for the first time. In the presence of an excess of the corresponding 5,7‐dienes (ergosterol or 7‐dehydrocholesterol), these photoactive sterols constitute a self‐regenerating source of singlet molecular oxygen (1O2) during irradiation in vivo or in vitro with UVA bT15‐400 nm). The quantum yield for photosensitization of 1O2 by 9‐DHE was estimated to be 0.09. The 1O2 is scavenged by the dienes and the rate constant for 1O2 quenching by ergosterol was found to be 1.2 times 107M‐1 s‐1 in methyl t‐butyl ether (MTBE). This scavenging ultimately leads to the production of 5,8‐endo‐peroxide and hydrogen peroxide. Photochemically induced superoxide radical was also produced on irradiation of sterol 5,7,9‐trienes and trapped with the spin trap 5,5‐dimeth‐yl‐1‐pyrroline W‐oxide (DMPO). The production of singlet oxygen, peroxides and radicals by the sterols may be significant in the cell damaging and tumor promoting action of UVA light on skin.

Generation of hydrogen peroxide by incidental metal ion-catalyzed autooxidation of glutathione

Autooxidation of reduced glutathione in 50 mM buffer at pH 7.9 is indetectably slow in the presence of 1 mM DETAPAC, EDTA, TET, or tripyridine, but passing buffer through Chelex resin was insufficient to remove traces of catalytically active metals. Production of hydrogen peroxide during glutathione autooxidation was catalyzed by traces of Fe+2 or Cu+2, and to a much lesser extent by Cu+1 and Ni+2, but not to a detectable extent by Na+1, K+1, Fe+3, Al+3, Cd+2, Zn+2, Ca+2, Mg+2, Mn+2, or Hg+2. Cysteine was a much better precursor for hydrogen peroxide production than were cysteine sulfinic or sulfonic acids. The chelators EGTA, NTA, bipyridine, dimethyl glyoxime, salicylate, and Desferal were ineffective at preventing autooxidation. EDDA and 8-hydroxyquinoline were partially effective. Catalase could completely prevent the accumulation of detectable H2O2, but superoxide dismutase was only slightly inhibitory. Hydroxyl radical and singlet oxygen quenching agents (mannitol and histidine) stimulated. A mechanism for the production of H2O2 during trace metal catalyzed oxidation of glutathione is proposed, involving glutathione-complexed metal and dissolved oxygen. Although a radical intermediate can not be ruled out, no radical initiated chain reaction is necessary.

Activation of nonspecific lipase (EC 3.1.1.-) by bile salts

The enzyme nonspecific lipase (EC 3.1.1.-) from rat pancreas has been isolated and its amino acid composition determined. The amino acid composition confirms more indirect evidence that nonspecific lipase is not the same enzyme as cholesteryl ester hydrolase. Activation of the enzymatic activity by bile salts has been studied by equilibrium dialysis, gel filtration, light scattering, circular dichroism and fluorescence polarization. The binding of bile salt by the enzyme is saturable and is associated with a conformational change. Upon binding cholate, the protein experiences a decrease in beta-structure with no significant change in alpha-helix content, an increase in apparent Stokes radius, a decrease in light scattering properties, and a slight decrease in polarization of the intrinsic tryptophan fluorescence. Attachment of bile salt is associated with decreased reactivity of essential sulfhydryl groups, but no detectable change in reactivity of amino groups. A change to a more nearly spherical shape upon binding bile salt would be consistent with the experimental observations, but the exact sites of binding remain uncertain.