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SUPEROXIDE DISMUTASES IN ACUTE OMDATIVE STRESS OF LUNGDERIVED CELLS ~~_Kpri~O~~~~~~~~~~~~i~~for

Newborn infants are exposed to higher oxygen tension ex utem than in utero. Furthermore, the treatment of premature newborn babies with underdeveloped lungs often necessitates respiratory therapy with high oxygen which can cause several problems including chronic lung disease. We used transformed human cell lines of bronchial (BEAS2B) or alveolar (A549) epithelial origin to study the oxidative damage due to different oxidants (95 96 oxygen, 1OOuM H&, and 1oouM xanthlle with 75mU/ml xanthlne oxldase) as well as antioxidatlve mecbanllms (intracellular MnSOD and CuZnSOD, lmh4 N-acetylcysteine (NAC)). h4nSOD basal activity &as twice as high in A549 as in BEAS- cells. ln an acute exposure (4 h), n&A levels for Mn!3OD increased markedly dth 95 % oxygen and moderately with xanthine oxidase in A549 cells, but HzO, did not cause any than es in h4nSOD mRNA levels. There was no clear increase in Ml& D mRNA levels in BEAS2B cells with any of the exposures. h4nSOD and CuZnSOD activities were unchanged in both cell lmes after all ex sures. NAC dii not have any effect on Mn!ZOD nor 012 r&Y D activities or mRNA levels. We conclude that upregulation of MnSOD mRNA, but not activity, occurs in response to increased intra- and extracellular superoxide radical production in A549 cells. Further experiments are needed to elucidate the effect of oxidative stress on protein synthesis and possible posttranslational modification of SODS.

THE MACROPHAGE IN LDL OXIDATION: FRIEND OR FOE? Anna Baoutina, Roger Dean, Wendy Jessup

The Heart Research Institute, Sydney, Australia

Oxidation of low density liljoprotein (LDL) in the arterial intima has been implicated in atherogenesis. It is likely that the cells present in deceloping lesions, in&ding macrophag&, participate in such oxidation, thus playing a potentially proatherogenic role. In this paper we demonstrate a potentially protective role of macrophages towards LDL by virtue of their capacity to degrade lipid peroxides in the lipoprotein. In vitro generated cholesteryl ester hydroperoxides (CEOOH) in LDL were stable in cell-free media, but were rapidly cleared in the presence of several types of macrophages cultured in media non-permissive for LDL oxidation. This process depended on cell number, but could not be attributed to the net lipoprotein uptake. Cell-mediated loss of CEOOH appeared to be metal ion-independent. Degradation of CEOOH was accompanied by accumulation of low, non stoichiometric amounts of the corresponding hydroxide and cholesteryl keto- octadecadienoate. Cell-conditioned medium possessed a ‘similar capacity to remove CEOOH as seen with cellular monolayers, suggesting that the activity is not an integral component of the cell, but is secreted from it. The majority of this activity of cell- conditioned medium was associated with its high-molecular weight fraction (> 1OkDa) and was facilitated by the presence of free thiol are-uos. The abilitv ofmacroohaees to dezrade CECOH was xor in &e case of LDL*depleted bf &tocoph&ol but was restoied by replenishment of LDL tocopherol. Together with our earlier reports 06 the macrophage ability to rapidly remove peroxides from oxidised amino acids. oeotides and oroteins as well as to selectivelv clear cholesterol 7$-h^ydroperox\de, pur new results providk evidence of the potential protective activity of the cell towards LDL by removing reactive peroxide groups in the lipoprotein.

SKELETAL MUSCLE VITAMIN E AND UBIQUINONE 9 RESPONSES TO TRAINING AND LIPOATE SUPPLEMRNTATION

Even moderateintensity physical exercise may induce oxidative stress, in which antioxkiant defense systems in the body can not compensate for increased production of pro-oxidants. Regular physical exercise has been shown to enhance antioxidant protection. Vitamin E and ubiquinols are lipid phase antioxidants. alpha-Lipoate (LA), is a natural antioxkiant that is thou

p t to contribute to the recycling of

vitamlnCandvltamin .Weexaminedthee&ctof8weeksof treadmill trainine and ti SUDDkIll~t&iDl’l in 9 wk old Wistar rats (n= 4.0). I-k&of the rats &re divided as training group and the remaining half served as controls. Trained and untrained animals w&further divided into LA and saline supplemented groups. vitamin E and ubiquinone 9 levels were signiBcantly elevated in aerobic red gastmcnemius muscle of endurance trained animals (PcU.O5 and PcO.001 respectively). The effect of endurance training on vitamin E levels of anaerobic supe&zial vastus lateralis muscle was not as remarbble as seen in aerobic red muscle. LA supplementation did not affect vitamin E or ublquinone 9 levels in skeletal muscle. These results indicate that endurance training enhances lipid phase antioxidant defenses, especially in aerobic red skeletal muscle.

Supported by Finnish Ministry of Education grant to CKS.

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Porphyrin Mimics of Superoxide Dismutase: The Correlation Between Activity and Redox Potential, and the Advantage of Isomers of MnTMPyp over FeTMPyP”. Ina Batinic-Haberle.* Ludmil Benov,* Peter Hambright’, and Irwin Fridovich,* *Dept. Biochem., Duke University Medical Center, Durham, NC 27710,‘Dept. Chem., Howard University, Washington, D.C. 20059.

The SOD-like activity of Fe(II1) and Mn(ll1) complexes of ortho. mera and para isomers of 5,10,15,20-tetrakis(&methylpyridyl) porphyrins were found to be directly proportional to their metal-centered redox potential. An increase in potential of 260 mV for Fe(Il1) or 143 mV for the Mn(II1) porphyrins leads to a ten-fold increase in rate constant of Oz. dismutation. The behavior of the isomeric Mn porphyrins is in accord with the Marcus equation for outer-sphere electron transfer, whereas the Fe porphyrins have substantial inner-sphere character. For both metals, the ortho isomers have higher redox potentials than the meta and ortho forms, and at a given potential, the SOD-like activity is higher for manganese than for iron porphyrins. In tivo all Fe porphyrins were toxic under both aerobic and anaerobic conditions, to both SODdeficient and SOD-proficient strains of Escherichiu colt. Only the ortho and mera isomers, MnTM-2-PyP” and MnTM-3-PyP”, protected SOD-deficient E. coli. In comparison with para, the ortho and mefa species are sterically hindered with respect to rotation of the pyridinium groups into co-planarity with the porphyrin ring, making them have less tendency to intercalate into nucleic acids, accounting for their lower toxicities. The Mn complexes were preferable to the Fe derivatives possibly due to their lower tendency to add ligands to the coordinated metal ion both above and below the porphyrin plane. Thus the high-spin iron porphyrins may interact with proteins by axial ligation (in accord with inner-sphere behavior), producing low-spin derivatives which are better oxidizing agents by -200 mV, as compared with the high-spin Fe(II1) reactants and the corresponding manganese analogues. Consequently, iron porphyrins may act as oxidants rather than as antioxidants.

OXYGEN ’ 9 8 S27