Oxidative stress following traumatic brain injury in rats.

BACKGROUND: Free radicals may be involved in the pathophysiology of traumatic brain injury (TBI) through oxidative damage of neurovascular structures. Endogenous antioxidants, such as ascorbate and alpha-tocopherol, may play a critical role in combating these oxidative reactions and their oxidized products can serve as an important index of oxidative stress. METHODS: We used electron spin resonance (ESR) spectroscopy and in vivo spin trapping (reaction of an organic compound with free radical species) to detect the possible generation of free radicals after TBI. Injury was inflicted by a weight drop technique over the head (5.7 kg-cm). Rats were intravenously infused with either 1 mL, 0.1 M of the spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN), or an equivalent volume of saline immediately before TBI or sham-injury. Animals were divided into four groups: (1) Group I: PBN-infused sham-injured, (2) Group II: PBN-infused injured, (3) Group III: saline-infused sham-injured, and (4) Group IV: saline-infused injured. Additional groups of saline-infused uninjured, saline-infused, and PBN-infused injured animals were used for histopathology. Sixty minutes after TBI or sham-injury, rats were again anesthetized and decapitated. The brains were removed within 1 minute, homogenized, and extracted for lipids. The extracts were analyzed by ESR spectroscopy. Brain ascorbic acid (AA) concentration was determined spectrophotometrically, using the ascorbate oxidase assay. RESULTS: No PBN spin adduct signals (indicating trapped free radical species) were visible 60 minutes after TBI. All groups of rats showed an ascorbyl free radical signal. The ascorbyl signal intensity (AI) was, however, significantly higher in the injured rats, while the brain (AA) was significantly reduced. In addition, the ratio of AI/AA, which eliminates the effect of variable ascorbate concentrations in the brain, was also significantly higher in the injured animals. CONCLUSIONS: We conclude that 60 minutes following TBI there was a significantly increased level of oxidative stress in the brain. This may reflect formation of free radical species with subsequent interaction with ascorbate (antioxidant) during the 60 minute period. The lack of PBN spin adduct signals 1 hour after TBI may indicate that free radical generation is time dependent and might be detectable earlier or later than the 60 minute period.

Glucose-dependent formation of free radical species in lens homogenate.

Free radicals are thought to be involved in the pathogenesis of many disease states such as diabetes, cataractogenesis, cancer, and aging. Involvement of free radicals in most of these disorders, especially diabetic cataractogenesis, however, is derived from indirect evidence only. The purpose of this study was to directly detect the free radicals generated in lens homogenate incubated under hyperglycemic conditions. We investigated the generation of free radicals in lens homogenates incubated with 5.5 or 50 mM glucose containing the spin trap 5,5'-dimethyl 1-pyroline-N-oxide. The spin adducts were identified by electron spin resonance (ESR) spectroscopy and modulation of the levels of the spin adducts was achieved by including various antioxidants and metal chelators. Increased ESR signals were observed in the homogenates incubated with 50 mM glucose compared to those incubated with 5.5 mM glucose. The results show that the principal spin adduct has hyperfine splitting constants of a(N) = 1.41 +/- 0.01 mt and a(N) = 2.02 +/- 0.02 mt, indicating that these signals are due to carbon-centered free radicals. Superoxide dismutase, catalase, Trolox, and diethylenetriamine pentaacetic acid inhibited the signals while EDTA enhanced them. Based upon the results obtained by the inclusion of various modulators, it is concluded that the generation of the free radicals is mediated by a metal-derived Fenton type reaction.

Lipid peroxidation in free skin grafts in rats.

OBJECTIVE: To evaluate accumulation of products of lipid peroxidation in free skin grafts in rats over 10 days after grafting. DESIGN: Prospective analysis. ANIMALS: 30 adult Sprague-Dawley rats. PROCEDURE: Free skin grafts were applied to 1 hemithorax of 30 rats (2 groups, n = 15/ group), the other hemithorax acting as a nongrafted control. Biopsy specimens were taken from the nongrafted side of all rats immediately before and from the nongrafted and grafted sides immediately after the procedure. Biopsy specimens of both sides of the thorax were performed on days 1, 3, and 7 after grafting in group-1 rats, and on days 2, 4, and 10 after grafting in group-2 rats. Thiobarbituric acid (TBA) analysis for malondialdehyde and other aldehydic products (TBA-reactive substances) was used to determine lipid peroxidation. Concentration of TBA-reactive substances was determined by absorbance spectrophotometry at a wavelength of 532 nm. RESULTS: Accumulation of products of lipid peroxidation, reflected by increase in absorbance, continued to increase over the 10 days of this study. Difference in absorption between the nongraft and graft biopsy specimens was significant (P = 0.0011). Absorbance on days 3 and 4 was significantly higher than control and day-0 values (P < or = 0.05). Absorbance on days 7 and 10 was significantly higher than control, day-0, and day-1 values (P < or = 0.05). Rate of increase in absorption was maximal at day 4 and rapidly decreased thereafter. CONCLUSION: Accumulation of lipid peroxidation products in skin grafts may be best explained by oxygen-derived free radical-induced injury attributable to postischemic reperfusion. Maximal rate of accumulation corresponded to the known time of graft recirculation and revascularization. CLINICAL RELEVANCE: Predictability of free radical damage may allow timely pharmacologic intervention to reduce radical formation and ameliorate effects of peroxidation. Such intervention may help increase free graft survival or mitigate the effects of vascular compromise to axial pattern flaps.

The kinetics of the oxidation of L-ascorbic acid by peroxynitrite.

Peroxynitrite [O = NOO-, oxoperoxonitrate(1-)bd is a strong oxidant that may be formed in vivo by the reaction of O2.- and NO(.). Oxoperoxonitrate(1-) reacts with molecules in aqueous acidic solutions via pathways that involve the highly reactive hydrogen oxoperonitrate either as an intermediate in a first-order reaction or as a reactive agent in a simple second-order reaction. ESR experiments show that hydrogen oxoperoxonitrate oxidizes monohydrogen L-ascorbate by one electron: when mixed at pH ca. 5 and passed through a flow cell within 0.1 s, the two-line ESR signal of the ascorbyl radical anion (aH = 0.18 T, g = 2.005) is observed. The overall stoichiometry of the reaction was 1 mol of ascorbate oxidized per mol of oxoperoxonitrate(1-) added. The kinetics of the reaction were studied over the pH range 4.0-7.5 by stopped-flow spectrometry. Hydrogen oxoperoxonitrate, observed between 300 and 350 nm, and the oxoperoxonitrate(1-) anion, at 302 nm, disappear faster than predicted for the first-order isomerization to NO3-. The rate increases from pH 4 to 5.8, and then decreases with increasing pH. The rate variation suggests a bimolecular reaction either between the oxoperoxonitrate(1-) anion and ascorbic acid or between hydrogen oxoperoxonitrate and the monohydrogen ascorbate anion. Although the two pathways are kinetically indistinguishable, the pKa values of ascorbic acid and hydrogen oxoperoxonitrate strongly suggest that the reacting species are hydrogen oxoperoxonitrate and monohydrogen ascorbate. The second-order rate constant for this reaction is 235 +/- 4 M-1s-1 at 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of intravenous administration of hydroxyethyl-starch-deferoxamine on oxygen-derived free radical generation in cancellous bone specimens obtained from dogs.

The ability of IV administered hydroxyethyl-starch-deferoxamine to attenuate radical production in freshly procured cancellous bone specimens was investigated, using spin-trapping and electron spin resonance (ESR) techniques. A core cancellous bone specimen 10 mm long and 5.6 mm in diameter was obtained, using aseptic technique, from the proximal portion of the humerus of 30 adult mixed-breed dogs. After procurement of the initial bone specimen, 10 dogs received a 10% solution of hydroxyethyl-starch-deferoxamine in 0.9% NaCl (50 mg/kg of body weight, IV), 10 dogs received an equivalent volume (5 ml/kg, IV) of a 10% solution of hydroxyethyl-starch in 0.9% NaCl, and 10 dogs received 0.9% saline solution (5 ml/kg, IV). A second core cancellous bone specimen was obtained from the contralateral humerus of each dog 45 minutes after treatment. All specimens were individually incubated in the spin trap alpha-phenyl-N-tert-butylnitrone in Eagle's minimum essential medium, at 26 C for 45 minutes, then were frozen at -20 C until they were prepared for analysis by ESR spectroscopy. Each specimen was thawed, homogenized, and extracted in a low-dielectric organic solvent prior to obtaining an ESR spectrum, which was analyzed for hyperfine splitting constants for radical identification. Each first-derivative spectrum was digitally double-integrated to obtain an area; these areas were used to compare intensities of the spin adducts. Difference in the area obtained before and after treatment for each dog was expressed as a ratio of that dog's pretreatment area ([pretreatment - posttreatment])/pretreatment).(ABSTRACT TRUNCATED AT 250 WORDS)

Investigation of oxygen-derived free radical generation in cancellous bone specimens obtained from dogs.

Generation of free radicals and the ability of various antioxidants to attenuate radical production in freshly procured cancellous bone specimens was investigated, using spin-trapping and electron spin resonance (ESR) techniques. Seven core cancellous bone specimens, 10 mm long and 7.9 mm in diameter, were obtained using aseptic technique, from the proximal portion of the humerus of 9 adult mixed-breed dogs. One core cancellous bone specimen from each dog was incubated in spin trap alpha-phenyl-N-tert-butylnitrone in Eagle's minimum essential medium and served as a control. The other 6 specimens from each dog were incubated in alpha-phenyl-N-tert-butylnitrone/Eagle's minimum essential medium plus 1 of the following antioxidants: superoxide dismutase, catalase, superoxide dismutase/catalase, indomethacin, allopurinol, or deferoxamine mesylate. All specimens were incubated at 26 C for 90 minutes, then frozen at -20 C until they were prepared for analysis by ESR spectroscopy. Each specimen was thawed, homogenized, and extracted in a low-dielectric organic solvent prior to obtaining an ESR spectrum which was analyzed for hyperfine splitting constants to identify radicals. Each first-derivative spectrum was digitally double-integrated to obtain an area: these areas were used to compare intensities of the spin. For each treatment group, the areas from the treated specimens were compared with the areas from the control specimens, using a paired t-test. Significance was accepted at P < or = 0.05. Spin adducts were detected in all cancellous bone specimens. Specimens incubated in deferoxamine (P = 0.0017) and superoxide dismutase/catalase (P = 0.0452) had significantly smaller areas than did control specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen consumption and oxyradical production from microsomal reduction of aqueous extracts of cigarette tar.

Aqueous extracts of cigarette tar (ACT) contain a quinone-hydroquinone-semiquinone system that undergoes autoxidation to produce superoxide and hydrogen peroxide. In this paper we ask whether the production of activated oxygen species from this quinone system can be enhanced via an NAD(P)H-dependent autoxidation in which electrons are donated from electron transport components of the microsomal system to electron acceptors in ACT such as quinones and/or metal chelates. Therefore, the ability of ACT to enhance the oxidation of NADPH, consumption of oxygen, and production of oxyradicals during microsomal electron transport has been investigated. Rates of NADPH oxidation and oxygen consumption are enhanced in a concentration-dependent manner by both aqueous and dimethyl sulfoxide (DMSO) extracts of tar over that observed in the presence of microsomes alone. Over the time course studied, neither NADPH oxidation nor oxygen consumption by tar extracts is observed in the absence of microsomes. Microsomal NADPH oxidation is enhanced by 1,6- and 1,3-benzo[a]pyrene quinones and by iron-EDTA; per unit weight, the enhancement by these compounds is similar to that of tar. NADPH oxidation in the presence of tar and absence of iron-EDTA is inhibited by about 15% by the addition of deferoxamine to the reaction mixture. Chrysene quinone enhances NADPH oxidation to a much greater extent than does either of the benzo[a]pyrene quinones or tar. Inhibition of tar-stimulated microsomal oxygen uptake by superoxide dismutase, stimulation of oxygen uptake by azide (an inhibitor of catalase), and the presence of spin-trappable superoxide anion radical all indicate the reduction of tar components to superoxide and hydrogen peroxide. The similar effects of tar on NADPH oxidation catalyzed either by microsomes or by purified NADPH-cytochrome P450 reductase suggest that cytochrome P450 reductase is an important locus of electron transfer from microsomes to components of cigarette tar extracts. These data suggest that the cytotoxic and mutagenic effects of smoking may be mediated by metabolic activation of quinones and, to a lesser extent, metal chelates in cigarette tar to oxyradicals.

Effect of deferoxamine-hydroxyethyl pentafraction starch on free, autogenous full-thickness skin grafts in dogs.

Free, autogenous, full-thickness skin grafting was performed on 10 dogs; 5 dogs were given an iron chelator, deferoxamine-10% hydroxyethyl pentafraction starch (DEF-HES; 50 mg/kg of body weight, IV), and 5 dogs were given 10% hydroxyethyl pentafraction starch (HES) in 0.9% saline solution (5 ml/kg, IV). The percentage of viable graft on day 10 was higher, but not significantly so, in DEF-HES-treated dogs (mean +/- SD, 72.6 +/- 24.8%; median 76.5%) than in HES-treated dogs (mean +/- SD, 46.7 +/- 34.3%; median, 48.8%). A trend for a positive correlation between the percentage of viable graft (on day 10) and the percentage of original graft area (on day 28) was observed in HES- and DEF-HES-treated dogs; this trend was significant in HES-treated dogs (P = 0.012). Both groups had significant positive correlation between percentage of viable graft on day 10 and percentage of haired skin on day 28 (HES, P = 0.000002; DEF-HES, P = 0.0148). A unique finding in DEF-HES treated dogs was the consistent observation of foamy macrophages in the dermis adjacent to the grafts, in deep subcutaneous tissue below the grafts, and in normal dermis.

Separation and spectral data of the six isomeric ozonides from methyl oleate.

The structures of the products obtained on ozonation of methyl oleate have been re-examined. The assignments for the six isomeric ozonides of methyl oleate have been made by 1H and 13C nuclear magnetic resonance (NMR), which were consistent with the retention times observed in high-performance liquid chromatography; the assignments were confirmed by mass and infrared spectroscopy. Two triplets for the ozonide ring protons of the cis and trans isomers in the normal (MOO1) and the two cross ozonides (MOO1 and MOO3) can be resolved by 400 MHz NMR. For MOO1 and MOO3, where the two ring carbons are equivalent, two peaks for the ring carbons of each cross ozonides are resolved in the 13C NMR spectra, one for the cis and one for the trans isomer. For MOO2, four peaks for the ring carbons are resolved in the 13C NMR spectra, two for the cis and two for the trans isomer.

Release of iron from ferritin by aqueous extracts of cigarette smoke.

This study demonstrates the ability of aqueous extracts of cigarette smoke to reduce iron and cause its release from ferritin. Superoxide dismutase (SOD) increases the rates of iron release with the less filtered smoke extracts, but has no effect on the rate of iron release caused by aqueous extracts of well-filtered gas-phase cigarette smoke. Faster rates of iron release are observed under anaerobic conditions, and the reducing power of the cigarette smoke extracts is prolonged when incubated in argon. Hydroquinone and catechol, two of the major polyhydroxybenzenes in cigarette smoke, increase in concentration in the smoke extracts as these are subjected to less filtration, and higher concentrations of polyhydroxybenzenes correlate with higher rates of iron release from ferritin. Concomitant with iron release, depletions of amino acids in ferritin are observed. Depletion of histidine is partially prevented by bathophenanthrolinedisulfonate and mannitol, while lysine and arginine depletions remain unaffected. These observations suggest that cigarette smoke components react directly with these amino acid residues in ferritin. Cigarette smoke induced release of iron could alter iron metabolism in the lungs of chronic smokers and contribute to the increase in the total oxidative burden on the lungs of smokers.

The inhibitory effect of extracts of cigarette tar on electron transport of mitochondria and submitochondrial particles.

Acetonitrile extracts of cigarette tar inhibit state 3 and state 4 respiration of intact mitochondria. Exposure of respiring submitochondrial particles to acetonitrile extracts of cigarette tar results in a dose-dependent inhibition of oxygen consumption and reduced nicotinamide adenine dinucleotide (NADH) oxidation. This inhibition was not due to a solvent effect since acetonitrile alone did not alter oxygen consumption or NADH oxidation. Intact mitochondria are less sensitive to extracts of tar than submitochondrial particles. The NADH-ubiquinone (Q) reductase complex is more sensitive to inhibition by tar extract than the succinate-Q reductase and cytochrome complexes. Nicotine or catechol did not inhibit respiration of intact mitochondria. Treatment of submitochondrial particles with cigarette tar results in the formation of hydroxyl radicals, detected by electron spin resonance (ESR) spin trapping. The ESR signal attributable to the hydroxyl radical spin adduct requires the presence of NADH and is completely abolished by catalase and to a lesser extent superoxide dismutase (SOD). Catalase and SOD did not protect the mitochondrial respiratory chain from inhibition by tar extract, indicating that the radicals detected by ESR spin trapping are not responsible for the inhibition of the electron transport. We propose that tar causes at least two effects: (1) Tar components interact with the electron transport chain and inhibit electron flow, and (2) tar components interact with the electron transport chain, ultimately to form hydroxyl radicals.

tert-Butyl hydroperoxide-induced radical production in rat liver mitochondria.

When rat liver mitochondria are treated with tert-butyl hydroperoxide (TBHP) in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), electron paramagnetic resonance (EPR) signals are detected attributable to spin adducts resulting from the trapping of methyl, tert-butoxyl, and tert-butylperoxyl radicals. The addition of respiratory substrate results in a 3- to 7.5-fold increase in the signal intensity of the DMPO/methyl adduct, no change in the signal intensity of the DMPO/tert-butoxyl adduct, and complete loss of the DMPO/tert-butylperoxyl adduct signal. The magnitude of increase of methyl radical production in the presence of respiratory substrate is related to the respiratory control ratio (RCR) of the mitochondrial preparation. In the presence of antimycin A, which blocks electron flow between cytochromes b and c1, no stimulation of methyl radical production is detected with respiratory substrate. Stimulation of methyl radical production by the addition of respiratory substrate is detected in cytochrome c-depleted mitochondria. A similar increase in methyl radical production is detected when ferrous cytochrome c is treated with TBHP in the presence of DMPO (as compared to when ferricytochrome c is used). These results indicate that TBHP is reduced directly by either cytochrome c1, cytochrome c, or by both of these electron transport chain components in mitochondria undergoing state 4 respiration.

Free radical generation in the brain precedes hyperbaric oxygen-induced convulsions.

We tested the hypothesis that hyperbaric oxygenation (HBO) generates free radicals in the brain before the onset of neurological manifestations of central nervous system (CNS) oxygen poisoning. Chronically cannulated, conscious rats were individually placed in a transparent pressure chamber and exposed to (1) 5 atmospheres absolute (ATA) oxygen for 15 min (n = 4); (2) 5 ATA oxygen for 30 min (n = 5), during which no visible convulsions occurred; (3) 5 ATA oxygen for 30 min with recurrent convulsions (n = 6); (4) 5 ATA oxygen until the appearance of the first visible convulsions (n = 5); (5) 4 ATA oxygen for 60 min during which no convulsions occurred (n = 5); and (6) 5 ATA air for 30 min (n = 5, controls). Immediately before compression, 1 mL of 0.1 M of alpha-phenyl-N-tert-butyl nitrone (PBN) was administered intravenously (iv) for spin trapping. At the termination of each experiment, rats were euthanized by pentobarbital iv and decompressed within 1 min. Brains were rapidly removed for preparation of lipid extracts (Folch). The presence of PBN spin adducts in the lipid extracts was examined by electron spin resonance (ESR) spectroscopy. ESR spectra from unconvulsed rats exposed to 5 ATA oxygen for 30 min revealed both oxygen-centered and carbon-centered PBN spin adducts in three of the five brains. One of the five rats in this group showed an ascorbyl signal in the ESR spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

The ozonation of unsaturated fatty acids: aldehydes and hydrogen peroxide as products and possible mediators of ozone toxicity.

The products of the reactions of ozone with aqueous emulsions of unsaturated fatty acids and with liposomes made from phosphatidylcholine esters were characterized. Ozonolysis of emulsions of methyl oleate yields approximately 1 mol of hydrogen peroxide and 2 mol of aldehydes per mole of ozone used and fatty acid reacted. That is, the net equation that occurs is RCH = CHR' + O3 + H2O----RCHO + R'CHO + H2O2 . Ozonolysis of emulsions of oleic, linoleic, linolenic, and arachidonic acids gives 1 mol of hydrogen peroxide per mole of ozone used. Only very low yields (less than 5%) of reducible materials other than hydrogen peroxide are observed, suggesting that the yields of organic peroxidic materials, including Criegee ozonides and lipid hydroperoxides, are small. Ozonolysis of rat erythrocyte ghost membranes and rat bronchoalveolar lavage also gives significant yields (about 50%) of hydrogen peroxide based on the moles of ozone consumed. Reactions of ozone with bovine serum albumin, glutathione, and glucose do not produce hydrogen peroxide, implying that the hydrogen peroxide formed during the ozonation of biological materials arises almost exclusively from ozone/olefin reactions. Hydrogen peroxide and aldehydes are suggested to be important mediators of the modifications observed in both the lung and extrapulmonary tissues when ozone is inhaled.

Chemical, physical, and toxicological characterization of fumes produced by heating tetrafluoroethene homopolymer and its copolymers with hexafluoropropene and perfluoro(propyl vinyl ether).

Submicrometer solid-particle (fume) aerosols with up to about 10(8) particles/cm3 are formed when dilute vapors of low molecular weight (MW) (ca. 5000-10,000) perfluoro polymers condense in air. The aerosol (e.g., 0.2 mg/m3) formed from these vapors is highly toxic to laboratory rats by inhalation (30-min exposure) when fresh but loses toxicity in minutes, apparently owing to coagulation. These fumes may result from (a) pyrolysis of tetrafluoroethene homopolymer or tetrafluoroethene-hexafluoropropene copolymer or (b) evaporation, at 200-320 degrees C, of isolated low MW polymer or low MW polymer present in perfluoro polymer resins. Some perfluoro polymer fume aerosols contain reactive functional groups, but others without detectable reactive groups are at least as toxic.

Aldehydes, hydrogen peroxide, and organic radicals as mediators of ozone toxicity.

It is generally agreed that unsaturated fatty acids (UFA) are an important class of target molecule for reaction with ozone when polluted air is inhaled. Most discussions have implicated the UFA in cell membranes, but lung lining fluids also contain fatty acids that are from 20 to 40% unsaturated. Since UFA in lung lining fluids exist in a highly aquated environment, ozonation would be expected to produce aldehydes and hydrogen peroxide, rather than the Criegee ozonide. In agreement with this expectation, we find that ozonations of emulsions of fatty acids containing from one to four double bonds give one mole of H2O2 for each mole of ozone reacted. Ozonation of oleic acid emulsions and dioleoyl phosphatidyl choline gives similar results. with two moles of aldehydes and one mole of H2O2 formed per mole of ozone reacted. The net reaction that occurs when ozone reacts with pulmonary lipids is suggested to be given by equation 1. [formula: see text]. From 5 to 10% yields of Criegee ozonides also appear to be formed. In addition, a direct reaction of unknown mechanism occurs between ozone and UFA in homogeneous organic solution, in homogeneous solutions in water, in aqueous emulsions, and in lipid bilayers to give organic radicals that can be spin trapped. These radicals are suggested to be responsible for initiating lipid peroxidation of polyunsaturated fatty acids. Thus, aldehydes, hydrogen peroxide, and directly produced organic radicals are suggested to be mediators of ozone-induced pathology.

Comparative mutagenicity of nitrofluoranthenes in Salmonella typhimurium TA98, TA98NR, and TA98/1,8DNP6.

The nitration of fluoranthene, one of the most abundant polycyclic aromatic hydrocarbons (PAH) in diesel fuels, occurs in the laboratory under either electrophilic or free-radical conditions to give nitro-PAH. 3-Nitrofluoranthene (3-NF) is the major product under electrophilic ionic conditions while 2-nitrofluoranthene (2-NF) is the major product under free-radical nitration conditions. The free-radical nitration of fluoranthene also yields 1,2- and 1,3-dinitrofluoranthene (1,2-DNF and 1,3-DNF). Nitration on the 3-position of fluoranthene enhances the mutagenic potency more strongly than on the 2-position. Thus, 3-NF is a more potent mutagen than 2-NF and 1,3-DNF is more potent than 1,2-DNF, an isomer with one near coplanar nitro group and one nitro group substantially out of plane with the fluoranthene skeleton, when tested against Salmonella typhimurium TA98, TA98NR, and TA98/1,8-DNP6. In addition, the activation of these dinitro-PAH to mutagens does not depend on the "classical nitroreductase" and/or O-acetylase, suggesting that they are activated via different pathways. Despite the fact that 3-NF and 1-phenyl-4-nitronaphthalene (1-P-4NN), a non-planar analog of 3-NF, have virtually identical reduction potentials, their mutagenic potencies differ by three orders of magnitude. This finding suggests that when nitro-PAH of varying steric requirements are compared, the reduction potential may not predict mutagenic potency as well as had been previously suggested.

Aqueous cigarette tar extracts damage human alpha-1-proteinase inhibitor.

The elastase inhibitory capacity (EIC) of human alpha-1-proteinase inhibitor (alpha 1PI) is severely compromised by aqueous cigarette tar extract (ACTE). An aqueous extract of the tar from two cigarettes causes a loss of EIC of at least 60% in 24 h at 37 degrees C (pH 7.4) and the damaging capability of the ACTE is retained for many hours. Hydrogen peroxide appears to be an essential component of the mechanism by which ACTE damages alpha 1 PI, since catalase substantially protects alpha 1PI from ACTE-mediated damage. Only mild protection is offered by 10 mM diethylenetriamine pentaacetic acid, indicating only a minor role for transition metal ions in the alpha 1PI-damaging process. Hydroxyl radicals are unlikely agents of alpha 1PI damage in the ACTE system, as judged from hydroxyl radical scavenger studies. Ascorbate and various thiols offer protection to different degrees, dependent on the incubation conditions. Of several amino acids tested, cysteine and methionine (but not methionine sulfoxide) are the only two that protect alpha 1PI. We suggest that components of cigarette smoke particulate matter extracted into the aqueous lung fluid environment may cause local deficiencies in alpha 1PI in smokers' lungs.

The mixture of aldehydes and hydrogen peroxide produced in the ozonation of dioleoyl phosphatidylcholine causes hemolysis of human red blood cells.

Dioleoyl phosphatidylcholine (PC) liposomes were ozonized and the ozonized liposomes were tested for their lytic potency on human red blood cells (RBC). Ozonation of PC liposomes generated approximately 1 mole equivalent of hydrogen peroxide (H2O2) and 2 mole equivalents of aldehydes, based on the moles of ozone consumed. The time necessary for 50% hemolysis induced by ozonized liposomes (a convenient measure of hemolytic activity) was found to depend on the extent of ozonation of the PC liposomes, indicating the formation and accumulation of hemolytic agents during ozonation. Hemolysis was also observed when RBC were incubated with nonanal, the expected product of the ozonation of oleic acid, the principle unsaturated fatty acid in the liposomes. Hydrogen peroxide, another product of PC ozonation, did not induce hemolysis; however, a combination of H2O2 and nonanal was significantly more hemolytic than nonanal alone. A ratio of 1:2 H2O2/nonanal (the ratio observed in the ozonized liposomes) provided hemolytic activity comparable to that observed with ozonized dioleoyl PC. Among different antioxidants tested, ascorbate, catalase, and glutathione peroxidase partially inhibited hemolysis induced by ozonized liposomes and by H2O2/nonanal mixtures, but they were not protective against the nonanal-induced hemolysis. Identification of H2O2 and aldehydes as cytotoxic chemical species generated from the ozonation of unsaturated fatty acids may have an important bearing on the in vivo toxicity of ozone on the lung as well as on extrapulmonary tissues.