Session 1: Superoxide and Superoxide Dismutases 417

1:21 SIMILARITIES BETWEEN MECHANI SUS OF SUPEROXIDE

PRODUCTION IN ULTRASOUND TREATED HUMAN SERUM AND IN

BALF FROM PATIENTS WITH CHRONIC BRONCHITIS

Silvano Pinamonti, Mariavittoria Muzzoli, Milvia C. Chicca, *Leonardo M. Fabbri, *Franc0 Ravenna,

*Alberta Papi, *Adalbert Ciaccia, Department of Evolutionary Biology and *Institute of Infectious and Respiratory Diseases, University of Ferrera, via Borsari 46, I-44100 Ferrara, Italy.

Normal human serum in vitro treated with pulsed ultrasound exhibits a clastonenic activity detectable

by fluorescent analysis of DNA unwinding (FADU) on DNA extracted from lymphocytes of healthy blood donors Experiments with oxygen radical scavengers show that this clastogenic activity is caused by oxygen radicals, mainly superoxide. Among serum components which could be responsible for superoxide production, serum lipoproteins undergoing lipid peroxidation are likely candidates. We tested in vitro by FADU the clastogenic activity of ultrasound treated HDL, LDL and “LDL upon lymphocyte DNA and found that VLDL caused significant DNA damage in vitro. OUT research group also found that BALF from patients with chronic bronchitis (CB-BALF) exhibited a clastogenic activity detectable by FADU which could be quenched by oxygen radical scavengers. Kinetics of cytochrome c reduction showed that CB-BALF could indeed produce superoxide. In our experimental conditions, the likely responsible for superoxide production in CB-BALF appeared xanthine oxidase with either purines or unsaturated aldehydes as a substrate. In CB-BALF, aldehydes could be produced by 1 ipid peroxidation of the phosphatidyl choline (PC) fraction of lung surfactant. We found that ultrasound treated PC exhibited a clastogenic activity detectable by FADU and also significantly reduced cytochrome c in presence of xanthine oxidase, therefore producing superoxide. Kinetics of uric acid in presence of uricase and CB-BALF supported the presence of xanthine oxidase in CB-BALF. All these data point out to interesting similarities between superoxide production in ultrasound treated serum (in vitro) and in CB-BALF (in viva). Both systems could involve xanthine oxidase and lipid peroxidation upon either PC or serum lipoproteins and end up by producing superoxide damage upon DNA.

I:23 A STUDY OF THE REACTION BETWEEN SUPEROXIDE AND HYPOCHLORITE IN ALKALINE SOLUTION Michael J. Thomas and Qirui Chen Department of Biochemistry, Wake Forest University Medical Center, The Bowman Gray School of Medicine, Winston-Salem, NC, 27157 USA

The reaction between superoxide and hypochlorite was reported by Long and Bielski (J. Am. Chem. Sot., 1980) and they suggested that hydroxyl radical might be formed under the reaction conditions. Since activated neutrophils generate large quantities of both superoxide and hypo- chlorite, it is possible that the reaction between superoxide and hypochlorite is a source of hydroxyl radical that would participate in the normal bactericidal activities of the neutrophil. To test for the formation of hydroxyl radical superoxide was mixed with hypochlorite in the presence of benzoate. The superoxide was from the radiation induced decomposition of water in a microwave driven plasma lamp. The oxygen saturated aqueous solutions contained pM amounts of EDTA, 1 to 10 mM sodium formate at pHs between 10 and 11.5. Sodium hypochlorite and benzoate were the best commercial grades. The reaction products were analyzed by HPLC and by GC/MS. Hydroxy and chloro adducts were detected at all of the ring positions, but addition to the ortho position was favored. The yields were variable, but the hydroxylated benzoates were favored by a factor of from 4 to 10 over the chlorobenzoates. The relative ratios for the o:m:p products were 1.0:0.6:0.4 for chloro and, 1.00:0.22:0.06 for hydroxy adducts. The results do not appear to be consistent with the exclusive formation of the hydroxyl radical.

OXIDATIVE MODIFICATION OF SUPEROXIDE I:22 DISMUTASE. PHENOMENON OF RESERVATION OF CATALYTIC ACTIVITY BY NEW STRUCTURES APPEARANCING AFTER OXIDATION Boris P. Sharonov, Irina V. Churilova Institute of Pure Biopreparations St.Petersburg, 197110, Russia

Durin strut ure f

action of oxidants on SOD its is strongly changed and appear

new mol.ecular, forms which possess the catalytic activity. g Mild (ph siological) condo ions of oxidation is c aracterized the reservation of activity, small dec- rease of NH - roups and fast loss of SH- groups. 0x1 2 ation of NH -groups results in decrease of total electric charge (by data of electrophoresis) and additionally decrease the local electric fields (by data of influence of ionic strength on the activity of oxidized protein) which control the influx of 0 into the cavity

contrast to oxidation of oxidation of SH- effect because S

roups have the

with oxidants “false” target for oxidants.

NITRIC OXIDE REQUIRES SUPEROXIDE TO EXERT 1:24 BACTERICIDAL ACTIVITY Luca Brunelli and Joseph S. Beckman, Department of Anesthesiology, The University of Alabama at Birmingham, Birmingham, Alabama, 35233 USA

It is still unclear whether nitric oxide (NO) is directly toxic because, both in viva and in vitro, secondary reactIons with the ubiquitous superoxide (02-) cannot be easily ruled out. NO and 02. will react at near diffusion limit (Huie and Padmala, Free Rad Res Comm 1993, in press) to form the potent oxidant peroxynitrite (ONOO). E.cob exposed to 1 mM NO for up to one hour in both aerobic and anaerobic conditions did not show decreased viability. However, ONOO- killing was proportional to its concentration with an LD50 of 0.25 mM after ten minutes of exposure. The latter finding is m agreement with previous reports (Zhu et al, Arch Biochem Biophys 298, 452; 1992). The decomposition of the sydnonimine SIN-l also produces ONOO- through the release of NO and 02.. SIN-1 killing was proportional to its concentration with an LD50 of 0.5 mM after one hour exposure. The bactericidal activity of SIN-1 was enhanced by 02., since exposure to 0.5 mM SIN-l plus pterin/xanthine oxidase (X0) resulted in 0.1% survival. Intzrcstingly, the addition of FeEL‘TA 13 the same amounts of SIN-1 and pterin/XO, resulted m almost complete protection. Pterm/XO alone or together with FeEDTA showed only a slight decrease in viability. Our results prove that NO alone is not toxic to E.coh. The presence of 02- is required for NO to exert bactericidal activity through the production of ONOO-. The release of 02- by SIN-1 is likely to be the limiting factor m ONOO- production. Furthermore, ONOO- exerts a much greater toxicity to Ecoli compared to Oz., hydrogen peroxide (H202) and hydroxyl radical (OH’). The protection exerted by FeEDTA can be due to decreased 02-because of Its funneling to OH’. Alternatively, Fe could react directly with NO, thus reducmg the other fundamental chemical species in ONOO- production.