Oxidative interactions of synthetic lung epithelial lining fluid with metal-containing particulate matter

Guobin Sun1, Kay Crissman2, Joel Norwood2, Judy Richards2, Ralph Slade2, and Gary E. Hatch2

1 Curriculum in Toxicology, The University of North Carolina at Chapel Hill, Chapel Hill 27599; and 2 Experimental Toxicology Division, National Health and Environment Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Epidemiology studies show association of morbidity and mortality with exposure to ambient air particulate matter (PM). Metals present in PM may catalyze oxidation of important lipids and proteins present in the lining of the respiratory tract. The present study investigated the PM-induced oxidation of human bronchoalveolar lavage (BAL) fluid (BALF) and synthetic lung epithelial lining fluid (sELF) through the measurement of oxygen incorporation and antioxidant depletion assays. Residual oil fly ash (ROFA), an emission source PM that contains ~10% by weight of soluble transition metals, was added (0-200 µg/ml) to BALF or sELF and exposed to 20% 18O2 (24°C, 4 h). Oxygen incorporation was quantified as excess 18O in the dried samples after incubation. BALF and diluted sELF yielded similar results. Oxygen incorporation was increased by ROFA addition and was enhanced by ascorbic acid (AA) and mixtures of AA and glutathione (GSH). AA depletion, but not depletion of GSH or uric acid, occurred in parallel with oxygen incorporation. AA became inhibitory to oxygen incorporation when it was present in high enough concentrations that it was not depleted by ROFA. Physiological and higher concentrations of catalase, superoxide dismutase, and glutathione peroxidase had no effect on oxygen incorporation. Both protein and lipid were found to be targets for oxygen incorporation; however, lipid appeared to be necessary for protein oxygen incorporation to occur. Based on these findings, we predict that ROFA would initiate significant oxidation of lung lining fluids after in vivo exposure and that AA, GSH, and lipid concentrations of these fluids are important determinants of this oxidation.

autoxidation; residual oil fly ash; antioxidant; ascorbic acid; glutathione


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VARIOUS CLINICAL and epidemiological studies have associated increases in morbidity and mortality with ambient air concentrations of particulate matter (PM; see Refs. 8, 35, 42). Many environmental PMs contain measurable concentrations of metals (41, 44). Transition metals might contribute to the toxic effects of PM through catalysis of oxidation (31). The ability of PM to induce oxidation in vitro has been suggested as a predictor of in vivo toxicity (16, 36). Residual oil fly ash (ROFA), an emission source particle that is released into the atmosphere from oil-fired power plants, has been used as an environmentally relevant PM in toxicity studies. It contains ~10% by weight of water-soluble Fe, Ni, and V and traces of other metals (Table 1; see Ref. 20). Exposure of humans to ROFA has been associated with acute lung injury and development of lung disease (7, 33). Evidence has accumulated that soluble metals are critical to the toxicity of ROFA (9, 13, 24, 36) and that part of the injury induced by ROFA might be a result of oxidative stress (10, 11, 14, 15, 24, 26). It has been reported that free radicals and aldehydes are generated after intratracheal instillation of ROFA in rats (24, 29). The mechanisms underlying these effects are not well understood.

                              
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Table 1.   Water-soluble components of ROFA

Inhaled PM first makes contact with lung epithelial lining fluid (ELF) and possibly chemically interacts with ELF components. ELF is composed of surfactant lipids, proteins, and antioxidants (19, 39), and it is believed to serve as the first line of defense against inhaled toxins and infective agents. ELF contains high concentrations of antioxidants, including ascorbic acid (AA), glutathione (GSH), uric acid (UA), and alpha -tocopherol (AT), and antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and GSH peroxidase (GPx). Human ELF can be obtained in its diluted form from saline lavage of the lungs, but methods for concentrating this fluid back to its original form are not yet available. To study the oxidative interactions between ELF and PM, and to determine the contributions of different components of ELF, we formulated a synthetic ELF (sELF) based on literature data and our own measured values of human bronchoalveolar lavage (BAL) fluid (BALF) (5, 6, 19, 34, 43). Because the oxidation of ELF may involve oxygen incorporation into ELF components and changes in antioxidant concentrations, we have attempted to examine both of these processes. The goals of this study were 1) to formulate a sELF and investigate physiologically relevant autoxidation (oxidation by air) that might occur in this fluid when exposed to ROFA through measurement of oxygen incorporation and changes in antioxidant concentrations and 2) to compare oxidations in sELF with those occurring in human BALFs. Oxygen incorporation into sELF components during autoxidation was measured using 18O-labeled oxygen gas (20% 18O2 in nitrogen) in place of air and an isotope ratio-mass spectrometer for detection. Our results show that ROFA can induce significant oxygen incorporation into sELF and human BALFs and that AA, GSH, and lipid are determinants of these processes.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Particles. ROFA particles were collected from the Southern Research Institute (Birmingham, AL) with a Teflon-coated fiberglass filter downstream from the cyclone of a power plant in Florida that was burning a low-sulfur no. 6 residual oil (20). The collection temperature was 250-300°C.

Chemicals. Albumin (BSA), lysozyme (chicken egg white), apotransferrin (human), GSH, UA, AT, CAT (bovine liver), SOD (bovine erythrocytes), GPx (bovine erythrocytes), and Hanks' balanced salt solution without phenol red (HBSS) were obtained from Sigma Chemical (St. Louis, MO). Phosphatidylcholine (egg) was obtained from Avanti Polar Lipids (Alabaster, AL). AA was obtained from Aldrich Chemical (Milwaukee, WI). 18O2 gas (>95% isotopic purity) was obtained from Isotec (Miamisburg, OH). All chemicals were reagent grade or of higher purity.

Preparation of sELF. sELF was formulated (Table 2) based on literature data and our own measured values of human BALF (5, 6, 19, 34, 43). Apotransferrin was used as a surrogate for lactoferrin because it has similar iron-binding qualities yet is more readily available. Egg phosphatidylcholine was chosen as the lipid constituent of ELF because it contains a natural mixture of several esterified fatty acids, and its percentage of unsaturated lipids appears to be similar to that of human ELF (personal communication, Dr. S. Young, Duke University, Durham, NC). We also prepared sELF containing individual or different combinations of antioxidants (Tables 3 and 4) to determine their effects on oxidation. The following is the procedure for preparation of 100 ml of complete sELF (sELF containing all the constituents except antioxidant enzymes).

                              
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Table 2.   Constituents of synthetic lung epithelial lining fluid


                              
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Table 3.   Effects of antioxidant substances on autoxidation of sELF


                              
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Table 4.   Effects of antioxidant enzymes on autoxidation of sELF

A solution of phosphatidylcholine in chloroform (40 mg/ml, 25 ml) was added to a 100-ml glass tube followed by addition of 1.0 ml of AT in chloroform (0.1 mg/ml). The mixture was evaporated under nitrogen at room temperature. Next, 50 ml of HBSS were added, and the mixture was ultrasonicated in a water-ice bath until milky. To this milky mixture of lipids, a solution of proteins was added slowly (740 mg of albumin, 240 mg of lysozyme, and 20 mg of apotransferrin in 30 ml of HBSS). Finally, UA (0.5 mg/ml in HBSS, 5.0 ml), AA (5 mg/ml in water, 1.0 ml), and GSH (5 mg/ml in water, 1.0 ml) were added, and HBSS was added to a final volume of 100 ml. The sELF was adjusted to pH 7.4 using NaOH (0.2 M) and H3PO4 (0.2 M).

Other sELFs (Tables 3 and 4) were prepared following the same procedure with or without addition of specific reagent(s). The sELFs were stored at -80°C.

BAL procedure. Human BAL was performed as described previously (27). Six lavages for each person were pooled, and recovery of the injected saline was ~75%. The supernatants of the pooled lavages were stored at -80°C until assessments were made.

Reaction of sELF with 20% 18O2. Reactions of sELF with 20% 18O2 in nitrogen were conducted using a 96-well microtiter plate in a 1-liter plastic zipper bag. Each well had a diameter of 6.4 mm, giving an exposed surface area of 32.2 mm2. Briefly, a certain amount of sELF (190 µl, unless otherwise noted) was added to a microtiter plate followed by addition of other reagents as needed. ROFA suspension was added last (to final volume of 200 µl unless otherwise noted). Each well of the mixture was stirred with a pipette tip after addition of ROFA. Next, the microtiter plate was exposed to 20% 18O2 in the plastic zipper bag at 24°C for 4 h (except for the time-course study). All samples were run in duplicate on the same microtiter plate and exposure or on another plate and exposure.

After the exposure to 20% 18O2, an aliquot (100 µl) of each sELF sample was frozen at -80°C. The aliquot was lyophilized before 18O analyses and was stored in a zipper bag at 4°C. These samples were stable for at least 6 mo at 4°C.

Reaction of BALF or diluted sELF with 20% 18O2. Reactions of BALF or diluted sELF with 20% 18O2 were conducted the same way as described above for sELF except for the following: 1) a 48-well microtiter plate was used, and each well had a diameter of 11 mm, giving an exposed surface area of 95 mm2, 2) 1.5 ml of BALF or diluted sELF were added to each well for exposure, and 3) all samples were run in triplicate on the same microtiter plate and exposure.

After the exposure to 20% 18O2, the triplicate samples were combined and frozen at -80°C. These samples were lyophilized before 18O analyses and stored in a zipper bag at 4°C.

Measurement of 18O incorporation into sELF, diluted sELF, or BALF components. Assay for excess 18O was accomplished as described previously (22). Briefly, an elemental analyzer first converted oxygen in the dried samples to CO and measured oxygen contents of the samples, next a column filled with I2O5 converted CO to CO2, and, finally, an isotope ratio-mass spectrometer measured the fractional abundance of 18O of the resulting CO2. The results of excess 18O in the corresponding samples are expressed as micrograms 18O per gram of dry weight (µg/g dry wt).

Assays of antioxidant substances in sELF. After the exposure to 20% 18O2, an aliquot (40 µl) of each sample was added to 1 ml of 3% (wt/vol) perchloric acid and vortexed. The samples were centrifuged at 20,000 g at 4°C for 20 min. The supernatants were stored at -80°C and used for assays of AA, UA, and GSH. AA and UA were assayed by HPLC with amperometric electrochemical detection (28). Total GSH was determined by enzymatic recycling in the presence of GSH reductase and 5,5'-dithiobis-(2-nitrobenzoic acid) with a COBAS FARA autoanalyzer (1). Oxidized GSH was measured similarly except that vinylpyridine was added to samples before perchloric acid to block all reduced GSH (1, 18). AT was measured by HPLC with electrochemical detection (46).

Assays of total protein and phospholipid. Total protein in BALF or diluted sELF was assayed by the Coomassie blue protein method (Bio-Rad, Richmond, CA) with BSA as standard. Phospholipid in BALF or diluted sELF was assayed by the Wako method (enzymatic colorimetric method; Wako Chemicals, Richmond, VA).

Statistics. Data are expressed as means ± SE. Differences between data were analyzed for significance by performing Student's t-test. The results were considered significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Time course and ROFA dose response in oxidation of sELF by 20% 18O2. The time course of 18O incorporation into sELF in the presence of ROFA (50 µg/ml) was examined with complete sELF that contained all of the previously specified antioxidant substances (AA, GSH, UA, and AT). As shown in Fig. 1, 18O incorporation was almost completed (>90%) within the first 4 h and increased slightly over the next 2 h.


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Fig. 1.   Time course of 18O incorporation into synthetic lung epithelial lining fluid (sELF) in the presence of residual oil fly ash (ROFA). Complete sELF containing ROFA (50 µg/ml) was exposed to 20% 18O2 at 24°C for the given times. Means ± SE (n = 4) are shown.

We examined the effect of ROFA dose on 18O incorporation into sELF. Figure 2A shows 18O assay results of the ROFA dose response in 20% 18O2 in complete sELF and antioxidant-deficient sELF. ROFA had a large effect on 18O incorporation in complete sELF but had no significant effect on antioxidant-deficient sELF, except at high ROFA concentrations. Figure 2B shows that AA remaining in complete sELF at the termination of exposure decreased with the increase of ROFA concentration. At ROFA concentrations higher than ~50 µg/ml, AA (also initially present at 50 µg/ml of sELF) was completely consumed. We measured total GSH and oxidized GSH concentrations at the termination of 4 h of exposure of sELF in the presence of 100 µg/ml of ROFA (Table 5). We found that neither total GSH nor oxidized GSH concentrations were affected by ROFA addition. The reduced form of GSH (total GSH - oxidized GSH), therefore, was also not affected by ROFA addition. AT was decreased ~35% by 100 µg/ml of ROFA at the termination of exposure. The concentration of UA was also not affected significantly by ROFA addition. We did not follow up on assay of AT and oxidized GSH in subsequent experiments for technical reasons.


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Fig. 2.   Effect of ROFA concentration on 18O incorporation into sELF and on ascorbic acid (AA) concentrations immediately after incubation. Complete sELF or antioxidant-deficient sELF (sELF-AOs) containing the indicated concentrations of ROFA were exposed to 20% 18O2 at 24°C for 4 h. A: effect of ROFA on 18O incorporation. B: effect of ROFA on AA concentrations. Means ± SE (n = 4) are shown. *P < 0.05 compared with corresponding sELF samples without ROFA.


                              
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Table 5.   Effects of ROFA on GSH and AT in autoxidation of sELF

Nature of 18O incorporation into sELF constituents. To determine whether 18O was incorporated into ROFA itself or into antioxidant substances present in the sELF mixture, control experiments in which ROFA (200 µg/ml) was added to all constituents of sELF except protein and lipid were performed. The same incubation time, temperature, and antioxidant concentrations were used. None of these experiments showed any detectable 18O incorporation. We also conducted the same experiments using complete sELF and ROFA. After exposure to 20% 18O2, the samples were dialyzed (molecular weight cutoff 1,000) for 24 h to remove small molecules such as antioxidant substances and their oxidation products. The 18O incorporation measured after dialysis was the same as that before dialysis. These results suggested that the antioxidant substances and ROFA itself did not contribute significantly to 18O incorporation and that the measured 18O incorporation was the result of incorporation into sELF lipids and/or proteins.

We prepared lipid-deficient sELF (sELF - lipid) and protein-deficient sELF (sELF - protein) to examine the relative amounts of 18O incorporation into protein vs. lipid in the presence of ROFA (Table 6). No 18O incorporation was detected in the absence of lipid. When only lipids were present, 18O incorporation was observed comparable to complete sELF. Next, we extracted sELF samples containing both lipid and protein with chloroform after incubation to remove lipid and lipid oxidation products. We found that ~30-50% of 18O contents remained after extraction. These results suggested that 18O incorporation into protein occurred in the presence of oxidized lipid.

                              
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Table 6.   Relative contribution of protein vs. lipid to 18O incorporation

Effects of antioxidant substances on ROFA-induced oxidation of sELF by 20% 18O2. We prepared sELF containing normal concentrations of individual or different combinations of antioxidant substances (AA, GSH, UA, and AT) to study their contribution to 18O incorporation into sELF (Table 3). AA had a different effect from the other antioxidants. At normal physiological concentrations, AA (50 µg/ml) acted as a prooxidant in the presence of ROFA, greatly enhancing 18O incorporation into sELF. GSH enhanced 18O incorporation only at the highest concentration of ROFA (200 µg/ml). UA and AT slightly and insignificantly enhanced 18O incorporation at the highest concentration of ROFA. AA alone appeared to enhance 18O incorporation more than the combination of the four antioxidant substances did. The 18O incorporation into sELF samples that contained AA were similar to each other in having high 18O incorporation. All sELF samples not containing AA were also similar to each other in having low 18O incorporation. These results indicated that the overall effect of the antioxidant substances of sELF was determined mainly by AA.

The measurement of antioxidants in the sELF at the termination of these exposures confirmed that AA was the only antioxidant that was depleted significantly after ROFA exposure (data not shown).

Effect of the ratio of AA to GSH on ROFA-induced oxidation of sELF by 20% 18O2. AA and GSH are water soluble and are present in ELF in relatively high concentrations. It has been reported that AA and GSH have actions in common and function together as part of a physiologically significant antioxidant system (23). Our preliminary experiments suggested a complex relationship between AA and GSH on 18O incorporation. Therefore, we altered concentrations of both AA and GSH in sELF in a 6 × 6 matrix design to examine the effect of the AA-to-GSH ratio on ROFA-induced oxidation of sELF. We conducted these experiments with the following three different ROFA concentrations: 40, 80, and 200 µg/ml. Figure 3A shows 18O incorporation for the samples that contained 200 µg/ml of ROFA. AA enhanced 18O incorporation in a dose-dependent manner at low to medium concentrations and then inhibited at high concentrations. Intermediate concentrations of AA led to the greatest 18O incorporation. GSH also enhanced 18O incorporation, but the GSH effect was not as great as that of AA. Figure 3B shows AA remaining in the same samples that contained 200 µg/ml of ROFA at the termination of exposure. AA was completely consumed at the termination of exposure if its concentration before exposure was lower than ~50 µg/ml. GSH had no significant effect on AA consumption. For the samples containing 40 or 80 µg/ml of ROFA, both oxygen incorporation and AA assay results (data not shown) followed the same patterns as that in Fig. 3 with the following differences: 1) 18O incorporation was lower than in the samples containing 200 µg/ml of ROFA, and 2) at lower ROFA concentrations, the consumption of AA occurred at lower AA concentrations.


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Fig. 3.   Effect of the ratio of AA to glutathione on ROFA-induced autoxidation of sELF. Three-dimensional graphs show data from a 6 × 6 matrix of microtiter wells containing sELF and ROFA (200 µg/ml) and exposed to 20% 18O2 at 24°C for 4 h. A: 18O incorporation. B: AA remaining at the termination of exposure. Note the biphasic nature of the 18O incorporation, with increases in incorporation as AA is increased from low values and then a diminishing of 18O incorporation at concentrations where higher and higher concentrations of AA remained in the incubation after exposure.

We also measured UA and total GSH concentrations in all of the above samples (data not shown). UA and total GSH were not depleted under any condition.

Effects of antioxidant enzymes on ROFA-induced oxidation of sELF by 20% 18O2. Based on antioxidant enzyme concentrations in normal human ELF obtained by Cantin et al. (5, 6), we prepared sELF containing individual or different combinations of these enzymes (CAT, SOD, and GPx; Table 4) in concentrations up to double the reported levels. Our interest was in determining actual physiologically relevant effects of these enzymes. The results indicated that the antioxidant enzymes at normal physiological concentrations (CAT: 4 U/ml, SOD: 40 U/ml, GPx: 0.05 U/ml) and at one-half or double these concentrations had no significant effect on ROFA-induced 18O incorporation into sELF. Even much higher concentrations of CAT and SOD (100 U/ml) had no significant effect. AA was completely consumed in the presence of 160 µg/ml of ROFA regardless of the presence of antioxidant enzymes (data not shown). UA and total GSH concentrations did not change in any of these samples.

Comparison of sELF with BALFs. We examined BALFs from five healthy people in both the presence and absence of added normal concentrations of AA (50 µg/ml). We also diluted sELF with saline solution to different degrees (50-, 100-, and 200-fold dilutions) to compare sELF with human BALFs. We measured total protein and phospholipid contents of BALF and diluted sELF. Table 7 shows that total protein contents of the BALF samples ranged from 57 to 116 µg/ml, approximately equal to the 100- to 200-fold diluted sELF, respectively (58.7-109 µg/ml). Phospholipid contents of the BALF samples ranged from 2.1 to 3.5 µg/ml, which was lower than even the 200-fold diluted sELF (7 µg/ml). We then measured 18O incorporation into BALF and diluted sELF in the presence and absence of ROFA (100 µg/ml) after exposure to 20% 18O2. ROFA caused significant 18O incorporation in both BALF and diluted sELF. Diluted sELF contained less incorporated 18O than what might have been expected based on multiplying the values observed in undiluted sELF (13,800 ng/ml) by dilution factors (1/50, 1/100, and 1/200). This suggested that greater oxidation might occur in native ELF than in BALF, which represents a large dilution of ELF. The 18O incorporation in both the BALF and diluted sELF samples was greatly enhanced by addition of AA (to 50 µg/ml to compensate for lowering of AA during dilution and loss during storage of BALFs). This increase in 18O incorporation induced by AA addition was observed in both the ROFA-containing samples and those without ROFA addition. The 18O incorporation into 200-fold diluted sELF was similar to that of the BALF samples.

                              
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Table 7.   Comparison between human BALFs and sELF


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of the present study was to formulate a sELF and to investigate oxidations that might occur in this fluid and in human BALF during exposure to PM that might be present in ambient air. Our eventual goals are to understand the role of PM-induced oxidation in PM toxicity and to develop an in vitro screening test that might predict PM toxicity. We also sought information on the usefulness of 18O-labeling techniques for detecting PM-induced oxidations and the role of antioxidant substances in these processes.

Our results showed that oxidation of sELF was easily detected as 18O incorporation after exposure to 20% 18O2. 18O incorporation was increased by ROFA and AA and slightly increased by GSH at some AA concentrations. Other antioxidant substances (UA and AT) known to be present in ELF had no significant effect either singly or in combination. ROFA concentrations as low as 20 µg/ml of sELF produced significant 18O incorporation. This concentration of ROFA is a reasonable approximation of possible human exposure. A person breathing 20 m3 of air containing a ROFA concentration of 150 µg/m3 (the national ambient air standard for PM of 10 µm or smaller; see Ref. 12) for 24 h and depositing 10% of the inhaled PM in a volume of 12.4 ml of airway and lung lining fluids (19) would have a ROFA concentration in the lining fluid of 24 µg/ml. If the ROFA were to be deposited in "hot spots" (the likely scenario), the concentrations might be much higher locally.

We compared our sELF with human BALF (Table 7). A 100- to 200-fold diluted sELF gave similar 18O incorporation to that observed in human BALFs. However, undiluted sELF incorporated more 18O than would be expected based on the diluted samples, suggesting that experiments with BALF might underestimate in vivo 18O incorporation. Replenishing AA that was lost during dilution increased 18O incorporation similarly in the BALF and the diluted sELF. The 200-fold diluted sELF was similar in protein concentration to human BALF samples; however, phospholipid concentrations were two- to threefold higher in diluted sELF than in the BALFs. When we determined the concentrations of lipid to use in sELF, we found that the ratio of phospholipid to protein varied greatly among species, with humans having the lowest ratio (43). Thus we maximized the lipid content of the sELF for initial studies to produce data relevant to both humans and animals and to increase chances for detecting oxidations. Human BALF is actually a mixture containing ~78% phospholipids and 22% neutral lipids (38). Because both types of lipids would be susceptible to oxidation, phospholipid measurements alone would underestimate true lipid substrate levels. We feel that we were successful in reaching our preliminary goal of synthesizing an ELF mixture and that further studies may build upon the present results. There is a need to extend these studies further to actual pulmonary surfactant lipids and proteins. Previous studies have also shown that oxidant production by macrophages might contribute to PM-induced oxidation (15, 21); thus, future studies will need to examine sELF in the presence of macrophages to more closely duplicate in vivo conditions.

In the present study, we found that little 18O incorporation into sELF occurred in the absence of ROFA, AA, or lipid. The ROFA-dependent 18O incorporation in the presence of both lipid and protein was higher than that in the presence of lipid alone. Removal of lipid from the oxidized mixture only partially reduced the apparent 18O incorporation, suggesting that some of the 18O incorporation was the result of an interaction between oxidized lipid and proteins. Previous studies in similar model systems support the notion that protein oxidation does not occur in the absence of lipids (3, 4, 17, 37, 45).

Measurement of the concentrations of antioxidant substances after incubation of sELF with ROFA provided increased insight into the oxidation processes. Only AA appeared to be depleted during the incubation. When concentrations of AA were high enough to be maintained throughout the incubation with ROFA, the enhancement in 18O incorporation was diminished (Fig. 3). Thus AA appeared to act as a prooxidant at low to medium concentrations, with antioxidant properties appearing at high concentrations. It has been reported that in Fe-dependent lipid peroxidation systems, it is the Fe2+-to-Fe3+ ratio that is necessary for lipid peroxidation to occur and that low concentrations of AA reduce some Fe3+ to Fe2+ and promote lipid peroxidation, whereas high concentrations of AA reduce too much Fe3+ to Fe2+ and inhibit lipid peroxidation (30, 32). ROFA contains a high content of water-soluble Fe (Table 1; see Ref. 20). Therefore, the biphasic property of AA in ROFA-induced 18O incorporation might be related to Fe-induced lipid peroxidation.

Antioxidant enzymes that have been reported to be present in ELF include CAT, SOD, and GPx (2, 5, 6). We found that these enzymes did not inhibit ROFA-induced 18O incorporation or loss of AA when added to sELF at physiological concentrations or at much higher concentrations. Published studies using other indicators of metal-catalyzed oxidation report inhibition by CAT in some studies but no inhibition in others (25, 40). Miller and Aust (30) reported that the rates of lipid peroxidation in the AA/Fe(III)/Fe(II) system were unaffected by the addition of CAT, SOD, or other hydroxyl radical scavengers. The lack of inhibition by these enzymes in our system might suggest 1) that superoxide and hydrogen peroxide are not involved in the oxidation by O2 and ROFA or 2) that metal binding by proteins and lipids causes oxidation to occur at such close proximity that the antioxidant enzymes are unable to intervene. Further studies will be necessary to resolve these issues.

The results of this study indicated for the first time that 18O labeling provided a useful measure of oxidation of sELF. 18O incorporation into sELF was measured easily after the incubation with 20% 18O2 in the presence of ROFA concentrations as low as 20 µg/ml. In a separate study, we report a comparison of this method with other methods of detecting oxidation, which were applied to the same samples (protein carbonyl formation, thiobarbituric acid-reactive substances, enzyme inhibition). The advantages of the 18O method over other methods are 1) it is more sensitive, 2) the oxidant source is unequivocal, and 3) it is possible to exclude contributions of oxidation occurring during sample preparation and color formation. We also report results of using individual metals and a bicarbonate, as opposed to phosphate, buffer system (unpublished observations).

In summary, 18O labeling methods and measurement of the loss of antioxidant substances provided assessments of oxidation of sELF and human BALFs induced by exposure to ROFA. AA (and to a lesser extent, GSH) enhanced oxidation, whereas other antioxidant substances and enzymes (at concentrations known to be present in ELF) did not affect oxidation. AA became inhibitory to oxidation at high concentrations. The presence of lipid was found to be necessary for protein oxidation to occur. Based on these findings, we predict that ROFA would initiate significant oxidation of lung ELFs after in vivo exposure and that AA, GSH, and lipid contents of these fluids are important determinants of these processes.


    ACKNOWLEDGEMENTS

We thank Drs. Dan Costa [Environmental Protection Agency (EPA)], Linda Birnbaum (EPA), David Warheit (DuPont Haskell Laboratories, Newark, DE), Maria Kadiiska (National Institute of Environmental Health Sciences), and Weiyi Su (Duke University Medical Center) for critical review of the manuscript and helpful comments and Shirley Henry and John McKee for technical assistance.


    FOOTNOTES

The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and the policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

This work was supported by Environmental Protection Agency/University of North Carolina Curriculum in Toxicology Grant 902908 (EPA-DuPont-Dow Cooperative Research and Development Award 0143-97).

Address for reprint requests and other correspondence: G. E. Hatch, Pulmonary Toxicology Branch, MD 82, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711 (E-mail: Hatch.gary{at}epa.gov).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 17 October 2000; accepted in final form 10 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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