341
Chem. Res. Toxicol. 1991,4,341-348
The Ozonation of Unsaturated Fatty Acids: Aldehydes and Hydrogen Peroxide as Products and Possible Mediators of Ozone Toxicity William A. Pryor,* Ballabh Das, and Daniel F. Church* Biodynamics Institute and Department of Chemistry, Louisiana S t a t e University, B a t o n Rouge, Louisiana 70803-1800 Received December 14,1990
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 RCHNHR’ + 03 + HzO RCHO + R’CHO + HzOz (1) +
Ozonolysis of emulsions of oleic, linoleic, linolenic, and arachidonic acids gives 1mol 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.
Introductlon Ozone is the most powerful oxidant to which humans are routinely exposed; it occurs at ppm levels in smog and presents major health problems for urban populations (1). The effects of ozone principally involve the pulmonary system (1-6),but extrapulmonary effects have also been reported in animals exposed to ozone (6-21). Ozone is too reactive to penetrate far into the air/tissue boundary in and the nonpulmonary the lung without reacting (12,231, effects of ozone must be due to subsequent reactions of the products that are formed from the reaction of ozone with primary target molecules. The nature of these products has remained unknown. There is a general consensus that unsaturated fatty acids (UFA)’are a primary target for ozone (1,5,6,14-22). This is quite reasonable for several reasons. First, while ozone reacts rapidly with many types of organic molecules, it reacts with olefins such as unsaturated fatty acids particularly rapidly (6,12-15,18,23,24). Second, the lungs of rats exposed to 2 ppm ozone in vivo show the presence of low molecular weight acids that clearly result from splitting small molecular fragments from unsaturated fatty acids by cleaving and oxidizing them at their double bonds, just as ozonation is known to do (25). Third, ozone is relatively lipophilic (13).And finally, vitamin E, which is largely confiied to the lipid bilayer in cells,protects both in vitro systems and animals against the effects of ozone (6). The chemistry of the reaction of ozone with olefins has been studied in detail (12,131.The great majority of these studies have utilized aprotic solvents, and in these solvents the Criegee ozonide is the major product and is stable (6, 12,26).Therefore, it is often assumed that the products
of the reactions of ozone with UFA in vivo are Criegee ozonides (26-28). We recently suggested that the UFA in lung lining fluids, including mucus and surfactant, are a primary target for ozone (29-31). Surfactant in different animals contains lipids with from 15% to 40% unsaturated fatty acids (32) and to some degree resembles an aqueous emulsion of fatty acids and other materials (33).If lung lining fluids are an important target for ozone, then the ozonation of unsaturated fatty acids in vivo occurs in the presence of water; this is significant since the ozonation of olefins in water gives carbonyl compounds and hydrogen peroxide, rather than Criegee ozonides. It also is noteworthy that even if the Criegee ozonides of fatty acids were formed, they are not very toxic (26,27,34) and probably cannot explain the nonpulmonary effects of ozone (6). The results described here indicate that ozonations of aqueous systems containing unsaturated fatty acids or their methyl or phosphatidylcholine esters produce approximately 1mol of hydrogen peroxide and 2 mol of aldehydes per mole of ozone reacted and of alkene consumed. Under our reaction conditions we observe the formation of very little (about 5% or less) other peroxidic materials (30,31).
* Address correspondence to these authors at the Biodynamics Institute, 711 Choppin, Louisiana State University, Baton Rduge, LA 70803-1800.
Abbreviations: DETAF’AC, diethylenetriaminepentaacetic acid; DNPH, 2,4-dinitrophenylhydrazine;EC, electrochemical;GSH, glutathione; PC, phosphatidylcholine; rbc, red blood cell(s); Tris, 2-amino2-(hydroxymethyl)-l,3-propanediol;UFA, unsaturated fatty acid(&
oa93-22a~/9i/2104-034i~02.5o/o
Materials and Methods Stearic acid (18:0),oleic acid (181),linoleic acid (18:2),linolenic acid (18:3),arachidonic acid (20:4), methyl oleate, methyl pentadecanoate, dioleoylphosphatidylcholine,soybean phosphatidylcholine, glutathione peroxidase, glutathione reductase, NADPH, glutathione (GSH), horseradish peroxidase, 3,3’,5,5’tetramethylbenzidine, bovine serum albumin (fatty acid free), g l u m , and diethylenetriaminepentaacetic acid (DETAPAC) were purchased from Sigma Chemical Co. (St. Louis, MO). Nonanal
1991 American Chemical Society
342 Chem. Res. Toxicol., Vol. 4, No. 3, 1991 and 5,5',7-indigotrisulfonic acid were obtained from Aldrich Chemicals. 2,4-Dinitrophenylhydrazine(DNPH) was purchased from Eastman Kodak. All the chemicals were used as received. Preparation of Rat Lavage and Erythrocyte Ghost Membranes. Lavage on rats was performed immediately following euthanasia, using 2 X 10 mL of a solution containing 0.145 M NaCl, 10 mM phosphate, and 0.2 mM DETAPAC, pH 7.4 (32). Lavage from two rata was pooled and centrifuged at 5OOg for 10 min to sediment cells, and the supernatant was used for the ozonation experiment. Blood from rata was collected into heparinized tubes by cardiac puncture. The blood was centrifuged at lOOOg for 10 min, plasma and buffy materials were aspirated out, and erythrocytes were washed 3X with 5 volumes of 10 mM phosphate containing 0.2 mM DETAPAC (pH 7.4). Washed erythrocytes were hemolyzed by the addition of 10 volumes of 10 mM Tris-HC1 containing 0.2 mM DETAPAC (pH 8.0) and hemoglobin-free ghost membranes obtained by successive washings and centrifugation at loooOg for 30 min, similar to the method described by Burton et al. (35). Finally, membranes were suspended in 10 mM phosphate/0.2 mM DETAPAC, pH 7.4, to the desired protein concentration for use. Protein was determined by the dye-binding method of Bradford (36) using bovine serum albumin as a standard. Preparation of Liposomes and Fatty Acid Emulsions. Liposomes of soybean phosphatidylcholine (PC) and dioleoyl-PC were prepared as described previously (24).Briefly, PC solutions in chloroform were dried in a rotary evaporator a t ambient temperature to obtain a thin film of lipids. A solution of 10 mM phosphate/0.2 mM DETAPAC, pH 7.6, was added, and the film was slowly peeled off to obtain a milky suspension of a desired concentration of liposomes. The suspension was subjected to sonication in an ice bath using a Branson 450 soflier at fullpower and a 50% duty cycle for 2 min. The sonication was repeated twice more at l0-min intervals, and the resulting solution was then allowed to stand in ice for a t least 1 h before use. Emulsions of fatty acids were prepared by dispersing fatty acids in 10 mM phosphate/O.2 mM DETAPAC, pH 7.6, and sonicating the solution for 10 min in a water bath sonicator (L & R Transistor/Ultrasonic T-14B). Ozone Generation. Ozone was generated by passing dried air through an ozonator (Welsbach Corp., Model T-23) at a fixed voltage and flow rate. The rate of ozone generation was determined by iodometry as described by us previously (14,15,18,=), by an indigo bleaching method (37), and by the UV absorption method (38). For the iodometric method, a 10% KI solution was bubbled with ozone in air for 1-3 min and the amount of Isliberated was determined spectrophotometrically a t 352 nm (t = 25.6 mM-' cm-').Similarly, for the indigo method, an air-ozone mixture was bubbled through a solution of 1mM indigotrisulfonic acid in 20 mM phosphoric acid for 1min, and the amount of ozone reacted with the dye was calculated from the decrease in absorption at 600 nm as compared to control (unozonized dye) by using a molar absorptivity value of 20 mM-' cm-'. For the UV absorption method, an air-ozone mixture was bubbled through acidified water (pH 2.0), and the amount of ozone dissolved in the solution was determined by analyzing the solution at 260 nm (e = 2.9 mM-' cm-'). Ozonation of various fatty acid emulsions, liposomes, rat erythrocyte ghost membranes, and rat branchoalveolar lavage was carried out by bubbling a stream of ozone in air through a fine capillary into the solutions. In each experiment, a 10% solution of potassium iodide was used as a trap following the reaction mixture for the estimation of unreacted ozone. Determination of Hydrogen Peroxide and Lipid Hydroperoxides. Hydrogen peroxide and lipid hydroperoxides were determined by three different methods as described below: (a) Glutathione Peroxidasdlutathione Reductase Coupled Enzyme Assay. Determination of peroxides by a glutathione peroxidase-glutathione reductase coupled enzyme system was performed by the end-point analysis as described by Heath and Tappel (39). The reaction mixture contained 0.02 unit of glutathione peroxidase, 0.08 unit of glutathione reductase, 0.1 mM NADPH, 1.0 mM GSH, and an appropriate amount (20-50 pL) of the ozonized sample in a 50 mM potassium phosphate buffer, pH 7.6, containing 1mM DETAPAC, in a final volume of 1mL. The decrease in absorbance at 340 nm was measured on a Hewlett
Pryor et al. Packard H P 8451A diode array spectrophotometer against a control sample that contained all additives but was not ozonized. The sum of the hydrogen peroxide and lipid hydroperoxide concentrations was determined from the net decrease in absorption at 340 nm by using an extinction coefficient of 6.23 mM-' cm-' for NADPH. The amount of hydrogen peroxide was determined by adding 200 units of catalase 4 min prior to the addition of GSH-peroxidaw the concentration of lipid hydroperoxide is taken as the total peroxide concentration without catalase minus the catalase-inhibitable peroxide concentration. The catalase-inhibitable peroxide values measured by this method are equal to the amount of hydrogen peroxide. (b) Horseradish Peroxidase Method. The method of Sznajder et al. (40) was used. The reaction mixture contained 0.15 M sodium acetate (pH 4.51, 0.1 mM 3,3',5,5'-tetramethylbenzidine, 10 units of horseradish peroxidase, and an appropriate amount (25-50 pL) of the ozonized sample in a final volume of 2.5 mL. The reaction was started by the addition of the enzyme. After 30 min of incubation a t ambient temperature, 0.5 mL of 10 N H2SOI was added, the reaction mixture mixed, and the absorbance of the samples measured at 450 nm against a control that contained an unozonized sample treated similarity. Concentrations of peroxides in the samples were calculated from a calibration curve obtained upon using known concentrations of hydrogen peroxide and tert-butyl hydroperoxide. As described above, sensitivity of the peroxide values to catalase was used to distinguish hydrogen peroxide and lipid hydroperoxides. (c) High-Performance Liquid Chromatography with Electrochemical Detection. For the electrochemical detection of hydrogen peroxide and lipid hydroperoxides in the ozonized fatty acid emulsions, a modified version of the method described by Yamada et al. (41) was used. A Perkin-Elmer Series 410 liquid chromatograph pump connected to a Perkin-Elmer LC-95 W/vis detector and ESA Coulochem (Model 5100A) electrochemical (EC)' detector was used. For analysis, 0.4 mL of the ozonized sample was mixed with 0.6 mL of methanol, and 0.02 mL was injected to a Hypersil ODS (5 pm, 100 x 2 mm) column, preequilibrated with 20% methanol and 80% water, a t a flow rate of 0.5 mL/min. The mobile phase contained 10 mM NaClO, throughout the sample analysis. The sample was eluted in 15 min with a linear gradient of 20-100% methanol. The eluent was monitored a t 234 nm for UV detection and at -400 mV for EC detection. Hydrogen peroxide and organic peroxides were distinguished by treatment of the sample with catalase, as described above, prior to analysis. Determination of Aldehydes. The method described by Lappin and Clark (42) was used to determine the total aldehyde concentrations in the ozonized fatty acid samples. Briefly, an appropriate amount ( 0 . 1 0 . 2 5 mL) of the sample was made up to 0.5 mL with water and mixed with 0.5 mL of methanol. To this mixture was added 1 mL of a saturated solution of 2,4-dinitrophenylhydrazine (DNPH) in methanol. After mixing the solution, 1drop of concentrated HC1 was added and the solution was incubated at 50 OC for 30 min. Tubes (16 X 1.8cm) containing the reaction mixture were covered with P a r a f i i to minimize loss of methanol due to evaporation during incubation. The contents were cooled in ice water, mixed with 5 mL of 10% KOH in 80% methanol, and analyzed at 480 nm within 5 min after the addition of alkali against a reference containing unozonized sample treated similarly. The concentration of aldehydes in the ozonized samples was calculated from a calibration curve created by using known amounts of nonanal (0.1-1.0 pmol). Gas Chromatographic Analysis of Methyl Oleate. Emulsions of methyl oleate were extracted 3X with chloroform/methanol(21). The extract was dried under nitrogen, and the residue was solubilized in 0.2 mL of hexane and analyzed on a Varian 3700 gas chromatograph equipped with a 100-m fused silica capillary SP-2560 column (0.25 mm i.d.) and a flame ionization detector. Untreated methyl oleate was used to construct a calibration curve, and methyl pentadecanoate served as an internal standard.
Results D e t e r m i n a t i o n of Ozone Concentration. Ozone concentrations in t h e aqueous reaction solutions and in t h e
Chem. Res. Toxicol., Vol. 4, No.3, 1991 343
Olefin Ozonations, Aldehydes, and Hydrogen Peroxide Table I. Production of Hydrogen Peroxide from the Ozonation of Unraturated Fatty Acidr and Liwsomesa sumed, method method fatty acid/liposome *mol Id 2' ND ND stearic acid emulsion 0.0 ND oleic acid in CClt 5.2 f 0.5 ND 4.0 f 0.2 3.6 f 0.4 3.8 f 0.2 oleic acid emulsion 4.6 f 0.2 4.5 f 0.4 4.4 f 0.1 linoleic acid emulsion 5.6 f 0.5 5.7 f 0.2 5.6 f 0.3 linolenic acid emulsion arachidonic acid emulsion 5.6 f 0.6 5.5 f 0.3 5.7 f 0.3 4.9 linoleic acid emulsionC 4.7 dioleoylphosphatidylcholine 4.3 f 0.2 3.5 f 0.2 3.7 f 0.3 liposome soybean phosphatidylcholine 5.0 f 0.1 4.5 f 0.1 4.6 f 0.3 liposome OThe concentration of fatty acids was 150 pmol in 30 mL of 10 mM phosphate/0.2 mM DETAPAC, pH 7.6. The ozonation of the reaction mixture was carried out for 15 min at an ozone delivery rate of 0.36 f 0.03 pmol in 305 mL of air per minute (26 f 2 ppm). ND, not detectable. Values are the average f SD of six replicates. bOleic acid (150 pmol) was dissolved in 30 mL of dry CCl,. 'A linoleic acid emulsion was ozonized for 2 h at an ozone delivery rate of 0.039 pmo1/(305 mL of air-min) (2.8 ppm). dGSH-peroxidase/GSH-reductase coupled m a y . e Horseradish peroxidase away.
trap solutions that followed them were determined by the three methods described above. The analysis of an acidified aqueous ozone solution for ozone concentration by the iodometry, indigo bleaching, and UV absorption methods gave values of 0.38 f 0.04,0.20 f 0.01, and 0.21 f 0.01 mM, respectively. Similarly, when exit gas was analyzed for ozone concentration by the iodometric and indigo bleaching methods, values of 0.65 f 0.03 and 0.35 f 0.03 pmol/min, respectively, were obtained. (The UV absorption method is not sensitive at these ozone concentrations.) These results indicate that iodometry overestimates the ozone concentration by a factor of 1.85as compared to the indigo bleaching and W absorption methods. (This value is considered accurate to 510% .) The iodometric method is nonstoichiometric,as is well-known (43,44),but it is fast and convenient, it has been frequently used by prior workers, and it is quite satisfactory as long as it is calibrated against a more accurate method. The ozone concentrations reported in the present study are based on the values obtained by the indigo bleaching and W absorption methods. On this basis, most of the experiments in our studies were performed at an ozone concentration of 26 f 2 ppm. A few experiments were also performed at 2.0 and 2.8 ppm ozone. Reaction of Ozone with Unsaturated Fatty Acid Emulsions/Liposomes and Formation of Hydrogen Peroxide. We have measured the yields of hydrogen peroxide from the ozonation of various fatty acids in dry organic solvents, aqueous emulsions, and liposomes as a function of time, the concentration of the fatty acids, and the concentration of ozone in the air stream. Production of hydrogen peroxide was confirmed by several methods, as discussed above. A comparison of the yields of hydrogen peroxide and the amount of ozone consumed by different fatty acid systems after 15 min of ozonation is shown in Table I. When oleic acid in CCl, was exposed to ozone, all of the ozone delivered to the solution was consumed, but no hydrogen peroxide was produced (Table I). Hydrogen peroxide also was not formed when an emulsion of oleic acid was exposed to air alone (data not shown). Stearic acid, a saturated fatty acid, did not consume ozone (Table 1).
4 I
-
3-
0
2
4
6
8
1
0
Time ( min )
Figure 1. Relationship between hydrogen peroxide and aldehyde generation during the ozonation of oleic acid emulsiona and dioleoylphosphatidylcholine liposomes. (A) Ozonation of 5 mL of a 2.5 mM oleic acid emulsion in 10 mM phosphate, pH 7.4. (B) Ozonation of 5 mL of 2 mM dioleoylphoaphatidylcholinelipoeomee in 10 mM phosphate, p H 7.4. Ozonations were for the indicated time periods a t the ozone delivery rate of 0.36 pmol and a flow rate of 305 mL of air per minute (26 f 2 ppm). Symbols: ( 0 ) hydrogen peroxide; ( 0 )aldehydes.
Ozonation of unsaturated fatty acid emulaions gave hydrogen peroxide yields that are approximately equivalent to the amount of ozone consumed (Table I). The yields of hydrogen peroxide determined by the GSHperoxidase/GSH-reductase or horseradish peroxidase assay are in excellent agreement. For liposomes, the yields of hydrogen peroxide also are approximately equal to the moles of ozone consumed (Table I). Lack of Substantial Yields of Other Peroxidic Products. After treatment with catalase, which destroys hydrogen peroxide but not lipid hydroperoxides, the ozone/olefin reaction mixtures were analyzed for lipid hydroperoxides by using the glutathione peroxidase or horseradish peroxidase assay. Neither analysis detected any lipid hydroperoxides in the ozone/olefin reaction mixtures. Analysis by HPLC with electrochemical detection of ozonated oleic acid emulsions revealed one principal peak at a retention time of approximately 1 min. Treatment of the ozonated sample with catalase prior to analysis caused complete disappearance of this peak, indicating that hydrogen peroxide is the principal peroxidic material. However, ozonated emulsions of linoleic acid gave small yields (less than about 5 mol % of the ozone delivered) of peroxidic substances other than hydrogen peroxide. Our preliminary data suggest that about 5% yields of Criegee ozonides are formed in these reactions. The hydroxy hydroperoxide would not be stable under our conditions, as discussed below (45). Relationship between Hydrogen Peroxide and Aldehyde Production. The relationship between the molar yields of hydrogen peroxide and aldehydes from the ozonolysis of oleic acid emulsions and dioleoylphosphatidylcholine liposomes is shown in Figure 1. The panel on the left shows the results of the ozonation of emulsions of oleic acid and that on the right dioleoylphasphatidylcholine liposomes; it is striking that the two systems give virtually identical yields of hydrogen peroxide and aldehydes per mole of ozone consumed. The data are consistent with a ratio of approximately 2 mol of aldehydes and 1 mol of hydrogen peroxide per mole of ozone consumed (Figure 1).
Ozonation of Unsaturated Fatty Acid Emulsions at Ozone Concentrations Nearing Smog Levels. Levels of ozone in smog occassionally reach levels as high as 0.5 ppm (1). It is important, therefore, to know whether the ozonation of fatty acid emulsions using ozone con-
344 Chem. Res. Toxicol., Vol. 4, No.3, 1991 12,
P
Aldehydes
Pryor et al.
-
Table 111. Production of Hydrogen Peroxide upon the Ozonolysis of Glucose, Protein, GSH, and Biological
(
Samples"
ozone consumed, HzO2, system pmol pmol 9i yieldb bronchoalveolar lavagec 1.08 f 0.20 0.57 f 0.03 55 erythrocyte ghost membraned 5.38f 0.20 2.08 f 0.18 39 bovine serum albumin (1 2.89 f 0.22 ND'
,
mg/mL)
GSH (100 pM) glucose (5 mM) 0
2
1
3
4
Time ( h ) Figure 2. Relationship between hydrogen peroxide and aldehyde generation during the ozonation of oleic acid emulsions with an air stream containing about 2 ppm ozone. The reaction volume contained 30 mL of 5 mM oleic acid in 10 mM phosphate, pH 7.4. Symbols: (0) hydrogen peroxide; ( 0 )aldehydes. Table 11. Relationships among Ozone Reacted, Methyl Oleate Consumed, and Hydrogen Peroxide and Aldehydes Formed time of ozonation, min 2.5 5.0 10.0
ozone
consumed,
oleate reacted,
pmol pmol 0.64 f 0.02 0.60 f 0.07 1.42 f 0.05 1.51 f 0.14 2.24 0.12 2.31 i 0.09
*
H20z,pmol
aldehydes,
pmol 0.59 f 0.01 1.18 f 0.07 1.34 f 0.04 2.55 i 0.39 2.15 f 0.29 4.15 f 0.28
" The values are the average f SD of three replicates. The concentration of fatty acid was 5 pmol in 5 mL of 10 mM phosphate, pH 7.4. The ozone delivery rate was 0.36f 0.03 pmol in 305 mL of air per minute (26 f 2 ppm).
centrations nearing smog levels exhibits reaction characteristics similar to that observed using the higher concentrations of ozone at which most of our studies were performed (near 26 ppm). When oleic acid emulsions were ozonated at ozone concentrations of approximately 2 ppm, a ratio of 1:2 between the yields of hydrogen peroxide and aldehydes was observed (Figure 2) and the amount of hydrogen peroxide produced was 1 mol per mole equivalent of ozone consumed. Linoleic acid emulsions upon ozonation at 2.8 ppm also yielded 1molar equivalent of hydrogen peroxide on the basis of the amount of ozone consumed (Table I). Stoichiometry of the Reaction of Ozone with Oleate. A comparison of the moles of ozone consumed, methyl oleate destroyed, and hydrogen peroxide and aldehydes produced upon ozonation of methyl oleate for different time periods is presented in Table 11. The data for 2.5 min of ozonation indicate that for each mole of ozone reacting with the oleate emulsions, approximately 1mol of oleate is destroyed and 1mol of hydrogen peroxide and 2 mol of aldehydes are produced. The data for 5-and 10-min ozonation show slightly lower yields of hydrogen peroxide and aldehydes based on oleate consumed. Ozonation of Rat Branchoalveolar Lavage, Erythrocyte Ghost Membranes, and Other Biomolecules. Table I11 shows the amount of ozone consumed and hydrogen peroxide produced upon the ozonolysis of rat erythrocyte ghost membranes, rat bronchoalveolar lavage, fatty acid free bovine serum albumin, glucose, and GSH. Ozonation of the lung lavage and ghost membranes resulted in yields of 40430% hydrogen peroxide based on ozone consumed. Although ozone reacted with BSA and GSH, no hydrogen peroxide was formed in these systems. Glucose did not react with ozone.
1.44 f 0.05 ND ND
0.0
"The values are the average f SD of three replicates. The reaction volume for bovine serum albumin, GSH, and glucose was 30 mL. The ozonation was carried out for 15 min at an ozone delivery rate of 0.36 f 0.03 pmol in 305 mL of air per minute (26f 2 ppm). *Based on ozone consumed. cConcentration of protein in lavage was 160 pg/mL, and the reaction volume was 10 mL. dConcentration of protein in ghost membranes was 320 pg/mL, and the reaction volume was 10 mL. ND, not detectable.
Dlscusslon Relationship between Ozone Consumed and Hydrogen Peroxide Formed. Our studies on the ozonation of unsaturated fatty acid emulsions show that virtually all of the ozone is accounted for by the production of hydrogen peroxide (see Table I). The data shown in Table I suggest that the yields of hydrogen peroxide from liposomes made from both natural and synthetic phosphatidylcholine esters may be only 85-90% on the basis of the ozone consumed, although Figure 1 indicates that the differences between fatty acid emulsions and liposomes made from synthetic PC must be small. The reasons for a lower hydrogen peroxide yield in liposomes are not known, but small amounts of ozone and/or hydrogen peroxide may react with phosphatidylcholine head groups or impurities in the PC. Measurement of Ozone Concentrations. It is important to recognize that the commonly used neutral KI method overestimates the concentration of ozone (4,18, 44,46). A recent critical review of the available methods for measuring ozone (4) concludes that the KI method is reproducible but is subject to 1040% variation depending upon the procedural variations and presence of interfering oxidizing or reducing substances and moisture in the gas stream. Recommendations therefore are made for using more than one procedure and the use of a correction factor for the KI method (4). The data presented here substantiate that conclusion, since the moles of ozone used agree with the moles of olefin used and hydrogen peroxide produced only if the KI method is corrected by a factor of 1.8. (See the discussion of this point under Materials and Methods). Mechanism of the Reaction of Ozone with Olefins in Aqueous Emulsions. Scheme I summarizes the reaction of ozone with olefins. Ozone reacts with olefins to produce a 1,2,3-trioxolane,2, that rapidly decomposes to form a carbonyl oxide, 3, and an aldehyde (6,12,13). The carbonyl oxide can subsequently react with an aldehyde to give the Criegee ozonide, 4, or with water to give a hydroxy hydroperoxide, 5 ( 6 , 1 2 , 13). The hydroxy hydroperoxide is metastable in aqueous solutions, hydrolyzing to give hydrogen peroxide and a second molecule of aldehyde (6, 12, 13). The formation of hydrogen peroxide from the ozonation of fatty acids requires carbon-carbon double bonds and water as a participating solvent. The saturated fatty acid, stearic acid, does not react with ozone, and the reaction of ozone with olefins in CCl, does not yield hydrogen peroxide (Table I). In aqueous solution, the rates of re-
Olefin Ozonations, Aldehydes] and Hydrogen Peroxide Scheme I. Mechanisms for the Reaction of Ozone with a lf-Disubstituted Olefin Such as an Unsaturated Fatty Acid in the Prewnce and Absence of Water 0
03
RCH=CHR’
1
I
RCH-CHR’
I
1 , 2 , 3-Moxohne
-
I
\
H
2
carbonyl
H
Criegee ozonide
oxide 3
5
4
action of a carbonyl oxide with water are faster than its reaction with aldehydes to form the Criegee ozonide (see Scheme I) (12,13,47‘). According to Scheme I, the reaction of ozone with unsaturated fatty acids in aqueous solutions should yield 1 mol equiv of hydrogen peroxide and 2 mol equiv of aldehydes for each mole of ozone and UFA consumed. That is, the net reaction is given by RCH==CHR’ + O3+ H20 RCHO + R’CHO + HzOz (1) Our results for fatty acid emulsions (Table I) and dioleoylphosphatidylcholineliposomes (Figure 1)fit this stoichiometry. The results of the ozonation of methyl oleate emulsions at 2.5 min show this stoichiometry; however, results at 5 and 10 min show 10-15% lower yields of hydrogen peroxide and aldehydes on the basis of ozone consumed. Criegee Ozonides, Hydroxy Hydroperoxides,and/or Lipid Hydroperoxides in Our System. Our studies detect hydrogen peroxide as the principal peroxidic material; the concentrations of hydroxy hydroperoxides and/or lipid hydroperoxides apparently are too low to be detected by the methods used here. However, in studies specifically aimed at detecting Criegee ozonides, we find that Criegee ozonides are formed in 1-5% yields when emulsions of unsaturated fatty acids or lipids (such as l-palmitoyl-2-oleoylphosphatidylcholine)in liposomes are exposed to ozone (M. Wu and U. M. Rao, unpublished work from these laboratories). It is also possible that small yields of lipid hydroperoxides are formed; we did not specifically search for them and did not detect them with the methods we used. We suggest that high yields of lipid hydroperoxides are not formed in our systems because lipid peroxidation is a relatively late event in the reactions of ozone with unsaturated fatty acid systems and the experiments reported here are done over short time periods. Hydroxy hydroperoxides are produced in ozone/olefin reactions that are carried out in aqueous solutions (see Scheme I), but we do not detect them. Hydroxy hydroperoxides decay to form aldehydes and hydrogen peroxide in a reaction that is general acid and general base catalyzed (45). Thus, the half-life of hydroxy hydroperoxides can be expected to be both solvent and pH dependent. To prepare and study hydroxy hydroperoxides, it is necessary to use high concentrations of hydrogen peroxide and/or aldehydes and/or to use a partially organic solvent. For
-
Chem. Res. Toxicol., Vol. 4, No. 3, 1991 345
example, Sander and Jencks (45)used 8 M HzOz,10 mM concentrations of various aldehydes,and a solvent that was 20% ethanol. Heath and Tappel studied the reaction of ozone with linoleic acid (39). They found that, in water at pH 6.8 (50 mM phosphate buffer), 53% of the ozone reacts to give hydrogen peroxide only] whereas in 90% ethanol, 71% of the ozone reacts to give a 35% yield of H20z and a 65% yield of an “organic hydroperoxide” [presumably the ethanol adduct of the carbonyl oxide, RCH(OCzH5)00H]. The reaction in which hydrogen peroxide and acetaldehyde are in equilibrium with the hydroxy hydroperoxide has an equilibrium constant equal to 50 M-l, and the half-life for decay of the hydroxy hydroperoxide at slightly acid pH values is of the order of seconds (45).Thus, hydroxy hydroperoxides that are intermediates in ozone/olefin reactions should not be observable under our conditions for several reasons: (i) Even with an equilibrium constant as large as 50 M-l, with both hydrogen peroxide and aldehydes a t millimolar levels as is true here, less than 1%of the aldehyde is present in the form of the hydroxy hydroperoxide at equilibrium, an amount too low for us to detect with our titration methods. (E.g., if the concentrations of both hydrogen peroxide and aldehyde equal M, the hydroxy hydroperoxide has an equilibrium concentration of 50 X 10” M.) (ii) At the near-neutral pH values we used, the rate of decay of the hydroxy hydroperoxide is very fast relative to the time scale of our experiments (&,a). (iii) Our solvent is water, where the hydroxy hydroperoxides are less stable. Reaction of Olefins with Ozone Concentrations Nearing Smog Levels. Most of our studies were conducted at Ozone concentrations of 26 ppm, which is higher than found in smog; levels of ozone in smog approach 0.5 ppm (1). However, ozonolysis of oleic acid emulsions a t the ozone delivery rate of 2 ppm also produced hydrogen peroxide and aldehydes in the ratio of 1:2, and the yield of hydrogen peroxide was 1 mol per mole of ozone consumed (Figure 2). Similar results were observed with linoleic acid emulsions (Table I). These results suggest that the reaction of lung lining fluid lipids with smog levels of ozone probably gives hydrogen peroxide and aldehydes as the principal products. Reaction of Ozone with Model Biological Systems and Formation of Hydrogen Peroxide. Hydrogen peroxide also was produced upon the ozonation of rat bronchoalveolar lavage and erythrocyte ghost membranes (Table 111). Yields of hydrogen peroxide (4040% on the basis of ozone consumed) in these systems were significantly lower than those observed from the ozonation of unsaturated fatty acid emulsions and liposomes. Lower yields of hydrogen peroxide from the ozonation of biological materials probably results from some of the ozone reacting with nonlipid components such as thiol-containing polypeptides and proteins; in fact, the rate constants for the reaction of ozone with thiols are significantly higher than with unsaturated fatty acids (23). Both BSA and GSH consumed a significant amount of ozone without the generation of detectable amounts of hydrogen peroxide, and glucose did not react with ozone (Table 111). Thus the source of hydrogen peroxide in the ozonation of lung lavage and erythrocyte ghost membranes appears to be virtually exclusively from the reaction of ozone with unsaturated fatty acids. Freeman and Mudd (49) have suggested that ozone reacts predominantly with proteins rather than with unsaturated fatty acids both in intact human erythrocytes and in human rbc ghosts. However, our studies on rat rbc ghost membranes indicate that ozone also must react with
346 Chem. Res. Toxicol., Vol. 4,No.3, 1991
erythrocyte unsaturated fatty acids, since hydrogen peroxide is produced in these reactions, and hydrogen peroxide appears to be a marker for ozone/olefin reactions. Elevated levels of hydrogen peroxide have been reported in red blood cells of rata and mice exposed to 5-7 ppm ozone on the basis of aminotriozole-inhibitablecatalase activity (50). Other Studies That Report Hydrogen Peroxide. Previous workers have not reported a one-to-one correspondence in the moles of ozone used and hydrogen peroxide produced (12,13,39,51,52). Tiege et al. (53)studied the lysis of rbc by ozonized egg PC liposomes; they found that ozonolysis of egg PC gave hydrogen peroxide in yields that averaged 46 f 5% on the basis of the ozone consumed. These data led them to conclude that 2 mol of ozone react to produce 1mol of hydrogen peroxide. They used the KI method for ozone without correction; if the correction factor of 1.85 is applied, their yields are 85%, in excellent agreement with our data on liposomes made from natural lipids. Heath and Tappel (39)report the ozonation of buffered solutions of linoleic acid and find a yield of ozone equal to 74% of the ozone used, they do not specify whether their KI method for ozone used a correction factor. Mudd et al. (54)ozonized spinach chloroplasts and obtained a 15% yield of total peroxides using an ~ n c 0 1 ~ e ~ t . d KI titration. They also observed that 2 mol of thiols were oxidized per mole of ozone used. Thiols are even more reactive toward ozone than are olefins (23),so it is likely that the thiols were directly oxidized by ozone rather than by a secondary product such as hydrogen peroxide. The reaction of most of the ozone with thiols, which according to our data does not produce hydrogen peroxide, undoubtedly explains the very low yield of hydrogen peroxide in this study (54). Mudd and Freeman in a review of ozone toxicology (55) conclude that ozonation of olefins produces hydrogen peroxide and hydroxy hydroperoxides. They comment that "It is possible that hydrogen peroxide or the hydroxyhydroperoxide could initiate radical reactions giving rise to lipid peroxidation, but this should be considered a side reaction". Our data support this suggestion, since we find that rbc lysis caused by ozonized PC is not radical-mediated but rather is caused by an unknown synergistic reaction of aldehydes and hydrogen peroxide (30,561. However, we do find that a direct reaction of ozone with olefins leads to spin-trappable free radicals and that this process occurs not only in organic solvents but also in homogeneous aqueous solution, micelles, and liposomes (30,31); it is likely that these organic radicals are at least partially responsible for the lipid peroxidation commonly observed when unsaturated fatty acids or lipids are exposed to ozone (30,31). Implications in Ozone Toxicity. Ozone is an extremely reactive molecule that is likely to react with the first reactive pulmonary constituents with which it collides. However, ozone-related biological alterations have been observed deep in lung cell layers (2-6)and at extrapulmonary sites (6-11). These observations suggest that the reaction of ozone with pulmonary constituents may produce toxic intermediate(s1 capable of passing through the air-blood barrier. A critical review of ozone toxicity (55) concludes the following: "A problem in elucidating the effects of ozone ...lies in determining whether ozone itself or some product released after the reaction of ozone with cellular constituents (such as H202,aldehydic compounds, free radicals, hydroperoxides) [is] responsible for the toxic effects".
Pryor et al.
The significant suggestion from our data is that olefins in the lung lining fluids and perhaps also in the membrane lipids of the cells immediately adjacent to the air boundary react with inhaled ozone to produce hydrogen peroxide and aldehydes. (a) Aldehydes as Toxins. Aldehydes are well-known toxicants and are stable enough to penetrate the pulmonary air-blood barrier and affect distant organs. Aldehydes arising from the oxidation of unsaturated fatty acids are known to damage functional proteins and nucleic acids and inhibit protein and nucleic acid syntheses (57-59). Aldehydes are also cytotoxic and capable of altering cellular immune functions (60-63). Kesner et al. (64,65) found that ozone-treatedphospholipids inhibit rbc ATPase and ascribed the inactivation to aldehydes; Freeman et al. (66)in a review of this work preferred to attribute these results to oxidation of amino acid residues by ozone. We have recently shown that ozone-treated phospholipids cause lysis of human rbc, and the effect can be duplicated by a 2:l mixture of nonanal and hydrogen peroxide (56). This 2:l mixture of nonanal/hydrogen peroxide is, of course, just what would be produced by the ozonolysis of oleate-containing PC. In 1965, Buell et al. (67)reported the exposure of rabbits to 1-5 ppm ozone; they extracted the lung tissue with ethanol and ethanol/ether, dried the combined solvents, and extracted the solid residue with heptane. The solids were hydrolyzed with proteases, and the hydrolysate was analyzed for aldehydes by using a dinitrophenylhydrazone assay. Several small aldehydes were identified, and the authors conclude that these aldehydes arise from ozonation of amino acid residues in collagen in the basement membrane (ethanal from alanine, 2-methylpropanal from valine, etc.) and that these aldehydes could cause cross-linking of proteins and alter lung structures (67). The identification of these small aldehydes is interesting. However, rate data suggest that amino acid residues such as valine are too unreactive toward ozone to be able to compete with olefins, the more reactive amino acid residues (such as cysteine), or potent electron donors (such as ascorbate) for direct reaction with ozone (14,23).In effect, olefins protect other materials from direct reaction with ozone (23). Furthermore, as discussed above, it seems doubtful that ozone itself could diffuse far enough into the lung tissue to be able to react with structural proteins such as collagen.
(b) Hydrogen Peroxide as a Toxin. Hydrogen peroxide also is a well-known oxidant in biological tissues, capable of damaging pulmonary endothelial cells (68)and directly inhibiting enzymatic functions by oxidizing important sulfhydryl groups on enzyme proteins (69).Hydrogen peroxide is readily converted to the more damaging hydroxyl radical by transition metals such as iron and copper (70).Recent studies implicate hydrogen peroxide as an important mediator of lung disorders, such as edema and adult respiratory distress syndrome (71,721,by impairment of the contractile properties of both distal and proximal smooth muscles (73)along with the inhibition of lung endothelial cyclooxygenase activity (74).Elevated levels of hydrogen peroxide are exhaled by humans suffering from acute respiratory failure (40). Summary and Conclusions. In summary, we find that ozonolysis of unsaturated fatty acid emulsions and liposomes produces approximately 1 mole equiv of hydrogen peroxide and 2 mol equiv of aldehydes, on the basis of ozone and alkene consumed (see eq 1). We also find that hydrogen peroxide is produced upon the ozonolysis of rat bronchoalveolar lavage and erythrocyte ghost membranes,
Olefin Ozonations, Aldehydes, and Hydrogen Peroxide
suggesting that unsaturated fatty acids are an important target for ozone attack in biological materials. Our results suggest that hydrogen peroxide and aldehydes generated upon the ozonation of unsaturated fatty acids present both in lung lining fluid and in cell membrane lipids may be important mediators of ozone toxicity in vivo.
Acknowledgment. This work was supported in part by a grant from NIH and a contract from the National Foundation for Cancer Research. Registry No. Os,10028-15-6; H202,7722-84-1; GSH,70-188; dioleoyl-PC, 4235-95-4;stearic acid, 57-11-4; oleic acid, 112-80-1; linoleic acid, 60-33-3; linolenic acid, 463-40-1; arachidonic acid, 506-32-1; glucose, 50-99-7; methyl oleate, 112-62-9.
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