Residual Oil Fly Ash Inhalation in Guinea Pigs: Influence of Absorbate and Glutathione Depletion

Joel Norwood, Jr., Alan D. Ledbetter, Donald L. Doerfler and Gary E. Hatch,1

National Health and Environmental Effects Research Laboratory, Office of Research and Development, MD-82, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Received September 29, 2000; accepted January 28, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhaled urban particulate matter (PM) often contains metals that appear to contribute to its toxicity. These particles first make contact with a thin layer of epithelial lining fluid in the respiratory tract. Antioxidants present in this fluid and in cells might be important susceptibility factors in PM toxicity. We investigated the role of ascorbic acid (C) and glutathione (GSH) as determinants of susceptibility to inhaled residual oil fly ash (ROFA) in guinea pigs (male, Hartley). Guinea pigs were divided into four groups, +C+GSH, +C–GSH, –C+GSH, and –C–GSH, and exposed to clean air or ROFA (< 2.5 micron diameter, 19–25 mg/m3 nose-only for 2.0 h). C and/or GSH were lowered by either feeding C-depleted diet (1 µg C/kg diet, 2 weeks) and/or by ip injection of a mixture of buthionine-S,R-sulfoximine (2.7 mmol/kg body weight) and diethylmaleate (1.2 mmol/kg, 2 h prior). Nasal lavage (NL) and bronchoalveolar lavage (BAL) fluid and cells were examined at 0 h and 24 h postexposure to ROFA. The C-deficient diet lowered C concentrations in BAL fluid and cells and in NL fluid by 90%, and the GSH-depletion regimen lowered both GSH and C in the BAL fluid and cells by 50%. ROFA deposition was calculated at time 0 from lung Ni levels to be 46 µg/g wet lung. In unexposed animals, the combined deficiency of C and GSH modified the cellular composition of cells recovered in lavage fluid, i.e., the increased number of eosinophils and macrophages in BAL fluid. ROFA inhalation increased lung injury in the –C–GSH group only (evidenced by increased BAL protein, LDH and neutrophils, and decreased BAL macrophages). ROFA exposure decreased C in BAL and NL at 0 h, and increased BAL C and GSH (2- to 4-fold above normal) at 24 h in nondepleted guinea pigs, but had no effect on C and GSH in depleted guinea pigs. Combined deficiency of C and GSH resulted in the highest macrophage and eosinophil counts of any group. GSH depletion was associated with increased BAL protein and LDH, increased numbers of BAL macrophages and eosinophils, and decreased rectal body temperatures. We conclude that combined deficiency of C and GSH increased susceptibility to inhaled ROFA; caused unusual BAL cellular changes; resulted in lower antioxidant concentrations in BAL than were observed with single deficiencies. Antioxidant deficiency may explain increased susceptibility to PM in elderly or diseased populations and may have important implications for extrapolating animal toxicity data to humans.

Key Words: inhalation; nose-only; guinea pigs; bronchoalveolar lavage; nasal lavage; ascorbic acid; uric acid; residual oil fly ash; glutathione; particulate matter..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidemiological data show associations between atmospheric particulate matter (PM) concentrations and adverse health effects in exposed populations (Spengler and Thurston, 1983Go; U.S. EPA, 1996Go). Evidence suggests that effects may be focused in the elderly or in other subpopulations with preexisting disease (Linn et al., 2000Go; Schwartz and Morris, 1995Go). Tissue concentrations of antioxidant substances such as ascorbate (C) and glutathione (GSH) appear to be lower in the elderly and in diseased individuals (Lenton et al., 2000Go; Rikans and Moore, 1988Go; Simon and Hudes, 1999Go).

Oxidant air pollutants (ozone, nitrogen dioxide) appear to exert greater acute injury in subjects depleted of antioxidants (Hatch et al., 1986Go; Slade et al., 1989Go). Dietary intake of certain antioxidants appears to offer some protection from these pollutants (Grievink et al., 1998Go); however, it is unclear whether dietary antioxidants might have a similar protective effect on exposure to PM. Some PM samples, such as residual oil fly ash (ROFA), contain a large percentage (10% by weight) of soluble metals iron (Fe), nickel (Ni), and vandium (V). Evidence from in vitro studies suggests that antioxidants may act as pro-oxidants in the presence of some of these metals (Fe, V) (reviewed in Stohs and Bagchi, 1995).

We hypothesized that antioxidant depletion might lower the toxicity of ROFA, because supplying fewer reductants to the metals might lower redox cycling of metals and the resulting oxidative effects. A previous study of intratracheally instilled silica in animals depleted of iron or ascorbate supported this idea (Ghio et al., 1998Go). Ghio and coworkers (2000) also reported similar effects of ROFA with respect to cytokine and inflammatory cell influx, lavage fluid protein, ascorbate, and lactate dehydrogenase (LDH) in wild-type and hypotransferrinemic mice. We also sought information on the influence of ROFA inhalation on antioxidant concentrations in nasal lavage (NL) and bronchoalveolar lavage (BAL) fluids. Previous investigators have suggested that toxicity of inhaled PM might be related to their ability to deplete antioxidants in respiratory tract lining fluids and cells (Zielinski et al., 1999Go).

Studies to date suggest that human lungs and BAL fluids contain considerably less (90% lower) C and GSH than rat and guinea pigs lungs or lung lavage cells (Slade et al., 1985Go, 1993Go). Thus, we reasoned that information on the combined deficiency might also be important for extrapolating inhalation toxicity data on PM from animals to humans. The effects of ROFA were examined by Dye and coworkers (1997) using rat tracheal epithelial cells. They showed that treatment resulted in cytotoxicity with depletion of GSH, and that buthionine-[S,R]-sulfoximine (BSO) augmented cytotoxicity. The same authors reported that decreased GSH levels, altered gene expression, cytokine production, and cytotoxicity were reversed by dimethylthiourea, suggesting a role for free radicals and oxidative stress. Similarly, Jiang and coworkers (2000) have reported the effects of several particulate matter samples on guinea pig tracheal epithelial cells. Other studies have described the histological features of lung injury induced by ROFA inhalation and intratracheal instillation and have demonstrated the significant role that metals play in its toxicity (Dreher et al., 1997Go; Kodavanti et al., 1997Go, 1999Go).

The goals of the present study were to investigate the effect of C and GSH depletion in guinea pigs on lung and nasal injury resulting from inhalation of ROFA; to determine whether ROFA inhalation alters antioxidant concentrations in nasal and lung lining fluids; and to obtain general information on the effect of a combined deficiency of C and GSH. Results show that acute ROFA inhalation produces adverse effects in guinea pigs deficient in both C and GSH that are not observed in singly deficient or normal animals.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Halothane was obtained from Aldrich Chemical Co. (Milwaukee, WI). BSO (> 97% pure) was purchased from Chemical Dynamics Corp. (S. Plainfield, NJ). Crystal violet; ethyl ether, anhydrous reagent; acetic acid, glacial; LeukoStat Diff-Quik staining solutions I and II; perchloric acid, 60% solution; saline, 0.9% sodium chloride solution; sodium hydroxide, pellets; and Tween-80 (70% oleic acid content) were purchased from Fisher Scientific Co. (Pittsburgh, PA). The Coomassie blue dye binding solution was purchased from Pierce Inc. (Rockford, IL). Adenosine 5'-triphosphate (ATP; 99%); ascorbic acid; bovine serum albumin (98% pure, fatty acid–free); diethylmaleate (DEM; 95% pure); EDTA, disodium salt (99% pure); firefly lantern extract-50; GSH (98% pure); Hank's balanced salt solution; LDH (reagent kit #228); 1,1,3,3-tetramethyoxypropane; 2-thiobarbituric acid (98% pure); trichloroacetic acid (99 % pure); tris-HCL (99.9% pure); and uric acid (99% pure) were purchased from Sigma Chemical Co. (St. Louis, MO).

Animals.
Specific pathogen–free Hartley strain guinea pigs (males, 350–450 g; 21 days old), obtained from Charles River Breeding Labs (Raleigh, NC), were used in all in vivo experiments. They were housed in American Association for Accreditation of Laboratory Animal Care (AAALAC)-accredited facilities in plastic cages with heat-treated wood chip bedding and kept in temperature- and humidity-controlled (72 ± 2°F, 50 ± 5% relative humidity) rooms with a 12-h light/dark cycle. Upon their arrival, animals were given food (Prolab Animal Diet, Agway Inc., Syracuse, NY) that consisted of guinea pig chow or rabbit chow and water ad libitum for 2 weeks prior to ROFA inhalation. HPLC analyses of C in the guinea pig chow and rabbit chow (Conventional Prolab Diet, Agway Inc., Syracuse, NY) indicated that rabbit chow was deficient in C (contains 1 µg C/kg diet) relative to the guinea pig chow (contains 2.34 mg C/kg diet).

Experimental design.
Three experiments were performed (Table 1Go). In the first, half of the guinea pigs were fed a diet that rendered them doubly deficient in antioxidants C and GSH (–C–GSH). The second experiment involved guinea pigs that were either GSH deficient only (+C–GSH) or C deficient only (–C+GSH). The third experiment involved all four guinea pig groups in the same experiment. Animals were randomized using a computer program that sorted the animals by weight, then assigned groups in such a way that resulted in similar mean body weights and standard deviations. All guinea pigs were acclimated to nose-only chambers for 1–2 h (24 h prior to the actual exposure).


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TABLE 1 Experimental Design
 
Body temperature measurement.
In some guinea pigs, rectal thermometer probes (RET-1 copper-constant thermocouple, Physitemp Instruments Inc., Clifton, NJ) were placed into the rectum prior to exposure, and body temperature was monitored (Omega Digital Thermal-Hygrometer, Model HR411, Omega Engineering, Stamford, CT) during exposure.

Depletion of tissue C and GSH levels.
Guinea pigs were fed rabbit chow, a diet lacking C, for 14 days prior to ROFA inhalation (Hatch et al., 1986Go). The remaining animals were fed a regular guinea pig diet for the equivalent duration. To lower tissue levels of GSH, a combination of 2.7 mmol BSO/kg body weight and 1.2 mmol DEM/kg body weight was injected (2–3 ml) ip 1–2 h prior to ROFA inhalation. The vehicle used was 15% Tween-80 in water. We showed previously that the use of DEM, in addition to GSH, is necessary to reduce lung GSH content acutely (Slade et al., 1989Go). Control guinea pigs were injected with an equivalent amount of vehicle only.

Exposure regimen.
ROFA was collected from a power plant burning low sulfur #6 residual oil on Teflon-coated fiberglass filters downstream of the cyclone at a collection temperature of 204°C (Southern Research Institute, Birmingham, AL). Previous assays showed that soluble Fe, Ni, and V make up about 10% of the weight of this ash (Hatch et al., 1985Go). Insoluble material made up about 10% of the weight.

A dust string generator (Ledbetter et al., 1998Go) was used that was capable of delivering an aerosol concentration of 0.5–50 mg/m3, with a total flow through the generator of 10–15 l/min. A particle charge neutralizer and a 2.5-µm cyclone was attached between the generator and the inhalation chamber to remove all ROFA particles larger than 2.5 µm in aerodynamic diameter. Actual chamber particle concentration was monitored gravimetrically once per hour during the exposure (RAM aerosol monitor, Model 1, GCA Corp., Bedford, MA). After ROFA inhalation, guinea pigs were anesthetized with 5% halothane (Frazer-Sweatman apparatus, Cyprane North America, Inc., Tonowanda, NY) immediately and 24 h later. While anesthetized, lungs were collapsed by puncturing the diaphragm. Exsanguination was assured by severing the abdominal aorta. All exposures were conducted at 8–10 a.m. and animals were killed at 10–12 a.m.

Determination of ROFA deposition into the lungs.
The cranial lobe of the whole lung (nonlavaged), air-, and ROFA-exposed animals, n = 4 in each group, were submitted blind for assessment of Ni and V content by neutron activation analysis (Nuclear Energy Services, Nuclear Engineering Department, N. C. State University, Raleigh, North Carolina).

Nasal lavage procedure.
After anesthesia and exsanguination, nasal cavities of guinea pigs were lavaged using a specially designed stainless steel plug inserted into the posterior pharynx through the mouth. The plug sealed off the oropharynx, forming a cup over the epiglottis, while providing a passage for saline to be injected through the posterior pharynx and out of the nares (Norwood et al., 1994Go).

The nasal cavity was lavaged with 2–3 ml 1.0 mM EDTA in 0.9% saline, pH 7.0, warmed to 37°C prior to injection and collected as it dripped from the nares into a tube. The NL fluid was kept briefly on ice and centrifuged (400 x g, 15 min, 5°C) to ensure that no cells and/or debris were present in the supernatant. Guinea pigs were fasted (all dietary groups) for 6.0 h (prior to ROFA exposure) to ensure that food particles in the mouth did not interfere with nasal lavage.

Lung lavage procedure.
Following NL, a stainless steel cannula was inserted into the trachea to about 0.5 cm above the carina and secured with a ligature. The whole lung was lavaged with 1.0 mM EDTA in 0.9% saline, pH 7.0, warmed to 37°C, in a volume equal to 30 ml/kg body weight. A single volume of saline was injected into the lungs and immediately withdrawn. The aspirated lung lavage fluid was reinjected into the lungs and withdrawn for a total of three times. The lung lavage fluid was placed in a test tube, kept briefly on ice, and centrifuged (400 x g, 15 min, 5°C) to separate the BAL cells from the BAL supernatant.

Preparation of lavage supernatants and cells.
Untreated NL and BAL supernatants and NL and BAL cells were analyzed for total protein and LDH content. NL and BAL cells were dissolved with 0.25N NaOH and stored until analyzed for total protein. NL and BAL cell suspensions were counted by hemacytometer following a 1:1 dilution with 0.1% crystal violet and 4% acetic acid in water (total cell count). Differential cell counts (about 200 total cells within the viewable frame) were determined by pelleting the NL and BAL cells (reconstituted with 0.4 ml of HBSS) onto cytocentrifuge slides (44 x g, 2 min) using a cytospin centrifuge (Shandon Inc., Pittsburgh, PA) and stained with a Diff-Quik (Fisher Scientific Co., Pittsburgh, PA) staining solution. At least 200 total cells were counted for each animal, and all cells within each high-power field were counted.

Perchloric acid (PCA, 60% solution) was added to NL and BAL supernatant to a final concentration of 3%. PCA (3%) was also added to NL and BAL cells. PCA-treated samples were stored at –80°C (found to be stable for at least 3 months) until ready for analyses. Prior to analyses, all PCA-treated samples were centrifuged (20,000 x g, 20 min, 5°C) and analyzed for C, UA, GSH, and nonprotein sulfhydryls (NPSH) content.

We have previously assayed for ATP in BAL fluids and cells as an indicator of toxicity, and thiobarbituric acid–reactive substances (TBARS) content was assayed as a crude index of oxidative stress. Trichloroacetic acid (TCA, 60% solution) was added to NL and BAL supernatants (to a final concentration of 6%), and a 6% solution was added to NL and BAL cell pellets immediately following centrifugation. Samples were stored at –80°C (stable for > 2 weeks). Prior to analyses, TCA-treated NL and BAL supernatant, and NL and BAL cells were centrifuged (20,000 x g, 20 min, 5°C), and the supernatants and cells were analyzed for ATP and TBARS content.

Biochemical assays.
The NL and BAL supernatant and NL and BAL cells were analyzed for total protein using the Coomassie blue binding method of Bradford (1976), using bovine serum albumin as a standard. LDH was assayed using Sigma Reagent Kit #228. The GSH assay (measured total GSH) was measured in PCA supernatants by the recycling method of Anderson (1985). NPSH, which measured all reduced SH groups and is mainly composed of GSH, was measured by the method of Sedlak and Lindsay (1968). These assays were run (on each separate animal in each group) on a COBAS FARA II centrifugal analyzer (Hoffman-La Roche, Branchburg, NJ), but the data were later pooled for statistical purposes.

The NL and BAL supernatant and NL and BAL cells were analyzed by HPLC for C and UA following the method of Kutnink et al. (1985), using reduced C and UA as standards. This method employed amperometric electrochemical detection.

The NL and BAL supernatant and NL and BAL cells were analyzed for TBARS following the methods of Sinnhubern et al. (1958) and Pryor et al. (1976). As a positive control, malonyldialdehyde (MDA) was measured in standard solutions using serially diluted concentrations of 1,1,3,3-tetramethyoxypropane. A solution of 1% wt/vol thiobarbituric acid (TBA) and 2.8% TCA were added to equal volumes of standards and to NL and BAL supernatant and to NL and BAL cells to measure for TBARS content. Equal volumes of TBA, TCA, and standard or unknown were mixed. After the heating step (100°C for 10 min), A532 was measured.

The NL and BAL supernatant and NL and BAL cells were analyzed for ATP following the methods of Gilles et al. (1976) and Spielmann et al. (1981). After extracting TCA from each standard and sample with 1.0 ml of ether, 0.125 ml of standards and samples were individually added to 1.0 ml of 50 mM tris-HCL. This was followed by the addition of 0.125 ml firefly lantern extract—50 to each vial. The luminescence detector was a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA).

Statistical analysis.
Data were analyzed using a four-way analysis of variance (ANOVA). The four independent variables were C (proficient or deficient), GSH (proficient or deficient), ROFA (present or not present), and time postexposure (0 h or 24 h). Each response variable was analyzed separately. Pairwise comparisons were performed as subtests of the overall model. Effects were judged to be significant if the p value was less than 0.05. No correction was applied to multiple comparisons and the reported p values are unadjusted.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of Antioxidant Depletion Regimens
The purpose of this study was to determine the effects of diminished C and GSH on injury produced by ROFA inhalation. Previous studies had shown that the regimens used to lower C and GSH effectively decreased whole-lung levels of C and GSH (Slade et al., 1989Go). The present study examined antioxidant changes in NL and BAL to confirm the antioxidant deficiencies, then examined BAL fluid indicators of injury. Table 2Go shows results of the ANOVA of the three main variables of the study: C depletion, GSH depletion, and ROFA exposure. Significantly increased values in the table are shown in bold, while significant decreases are shown in regular type. Numerical values shown in the table are factors that, when multiplied by the control value, equal the change observed relative to control.


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TABLE 2 Magnitude of Significant Changes in Analysis of Variance Results of Antioxidant Depletion and Residual Oil Fly Ash Inhalation in Guinea Pigs
 
The effectiveness of the C-depletion regimen was shown by the fact that C levels in NL and BAL were reduced to below 10% of control in animals on –C diet when pooled across time periods and at both 0 h and 24 h, respectively (Table 2Go). C levels in BAL cells were also decreased (to about 30% of control) in animals on –C diet (0- and 24-h pooled data and at 24 h). Other effects of the C-depletion regimen included the following: BAL and NL protein levels were increased (1.16–1.19x) and body weights were reduced (0.95x). C-deficient diet also increased BAL eosinophil counts at 24 h.

The effectiveness of the GSH-depletion regimen was shown by a loss of GSH from BAL (0.37x, 0 h) and from BAL cells (0.55x, pooled, and 24 h) (Table 2Go). The GSH-depletion regimen also had the following other effects: NL protein levels were increased (pooled and 24 h), NL C levels were decreased to 56% and 38% of control (pooled and 24 h), BAL cell UA levels were increased 3x at 24 h. NL TBARS were increased by GSH depletion when pooled across time periods. GSH depletion decreased BAL protein levels to 83% of control at 24 h. BAL LDH levels were increased by GSH depletion about 1.6x of control when pooled across time periods and at 24 h. GSH depletion increased BAL total cell counts (about 1.7x), BAL macrophages (about 1.8x), and BAL eosinophils (about 1.7x) when pooled across time periods and at 24 h.

Effects of ROFA Exposure on NL and BAL
Table 2Go shows that ROFA exposure decreased the percentage recovery of saline instilled during the BAL procedure (to 90% of control) when pooled across time periods. ROFA decreased NL C levels when pooled across time periods (to 37%) and at both 0 h (to 16%) and 24 h (to 53%) postexposure. NL UA levels were reduced by ROFA to 63% of control when pooled across time periods.

BAL fluid.
ROFA slightly increased BAL protein levels (1.2x) when pooled across time periods and at 24 h. BAL LDH levels were increased by ROFA at 24 h. BAL C levels were reduced by ROFA to 63% of control at 0 h. ROFA exposure increased BAL UA levels at 24 h. BAL GSH levels were significantly increased by ROFA exposure when pooled across time periods (1.7x) and at 24 h (2.5x). BAL total cell counts were decreased by ROFA exposure to 72% and 66% of control when pooled across time periods and at 24 h, respectively. Saline volume recovery was decreased to 90% at 24 h. BAL macrophage counts were decreased to 63% and 52% of control when pooled across time periods and at 24 h, respectively. ROFA exposure increased BAL neutrophil counts to 3.0x when pooled across time periods and at 24 h. BAL cell protein levels were increased by ROFA exposure to about 1.5x when pooled across time periods and 24 h. ROFA exposure decreased BAL cell UA levels to 13% of control at 24 h and increased BAL cell GSH levels (1.5x) at 0 h (Table 2Go).

To achieve a more detailed interpretation, selected results are explored by subtesting in Figures 1 and 2GoGo and in Table 3Go. As the main purpose of this study was to evaluate whether antioxidant depletion would affect ROFA toxicity, indicators of toxicity are shown in Figures 1 and 2GoGo at the 24-h time point, where they were most affected. It is clear that in general only one group of animals, those deficient in both C and GSH, showed significant increases in injury markers following exposure to ROFA. BAL protein levels were significantly increased by ROFA in both the –C+GSH and –C–GSH groups (Fig. 1AGo), whereas BAL LDH levels were increased only in the –C–GSH group (Fig. 1BGo). Figure 2AGo shows that ROFA exposure decreased BAL total cell counts in the –C–GSH group. This effect of ROFA was superimposed on an enhancing effect of the GSH and C depletion regimens alone on these measurements (as shown in Table 2Go). BAL macrophages in the –C–GSH group were decreased by ROFA to 39% of control at 24 h (Fig. 2BGo). BAL neutrophils in the –C–GSH group were increased by ROFA at 24 h (Fig. 2CGo), and BAL eosinophils were enhanced by C and GSH deficiency, but were not altered by ROFA inhalation (Fig. 2DGo).



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FIG. 1. Effects of residual oil fly ash inhalation (ROFA, 19–25 mg/m3 for 2 h; < 2.5 micron diameter) on guinea pig bronchoalveolar lavage fluid protein and lactate dehydrogenase. Guinea pigs were divided into four groups: normal (+C+GSH), glutathione deficient (+C–GSH), ascorbate deficient (–C+GSH), and doubly deficient (–C–GSH). Results are expressed as mean ± SE (n = 9) in comparison with its corresponding air-exposed group at 24 h postexposure. Asterisks denote a significant (p < 0.05) difference from air-exposed animals. Changes not associated with ROFA exposure are not shown in this figure, but are summarized in Table 2Go.

 


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FIG. 2. Bronchoalveolar lavage cell changes following inhalation of ROFA as described in the legend of Figure 1Go.

 

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TABLE 3 Subtests of Effects of Residual Oil Fly Ash on Nasal Lavage and Bronchoalveolar Lavage Ascorbate and Glutathione in Normal, Vitamin C-Deficient, and Glutathione-Deficient Guinea Pigs
 
Table 3Go shows data from individual groups of guinea pigs for some end points that demonstrated complex relationships. It is first apparent that the nose-only exposure to air induced some changes in the guinea pigs killed at 24 h postexposure. Particularly, BAL cellular C and GSH levels were increased. ROFA effects included a lowering of NL C in the +C+GSH and +C–GSH groups (to 0.11x and 0.03x, respectively) at 0 h. At 24 h, this effect was attenuated (to 0.46x) but still significant in the +C+GSH group. The BAL C level in the +C+GSH group was also reduced by ROFA to 0.56x at 0 h, but it was increased 1.9x higher than control 24 h later. ROFA increased BAL GSH levels in both the +C+GSH and the –C+GSH groups at 24 h, but had no effect in the GSH-depleted groups. Results of cellular concentrations of C and GSH indicate that the three following factors had a large influence.

Body Temperature Changes during Exposure
We included in the first two experiments animals that had been implanted with a rectal temperature probe. Previous studies suggested that changes in body temperature during exposure might signal breathing changes, which could impact on results observed (Slade et al., 1997Go). Table 4Go shows that some of the guinea pigs exhibited a hypothermic response during exposure. The rectal temperature of normal guinea pigs was about 39°C. Groups depleted in GSH (+C–GSH, –C–GSH) had rectal temperatures about 3° lower than normal. Neither ROFA exposure nor deficiency in C alone had an effect on body temperature.


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TABLE 4 Rectal Temperature Changes in Guinea Pigs with Antioxidant Alterations and Residual Oil Fly Ash Treatment
 
Metal Analysis of the Lung to Estimate Deposition of ROFA
Table 5Go shows the results of neutron activation analysis of lungs from ROFA-exposed animals. Analysis for Ni and vanadium V was done to confirm that ROFA actually reached the lung during the exposure and, if possible, to quantify the level of deposition. Ni and V were chosen as markers for ROFA because they are not normally present in high concentrations in the lung, and they each make up about 4% (3.7% for Ni and 4% for V) of the dry weight of the ROFA. Background levels of both Ni and V were measurable but low (< 0.3 µg/g wet). ROFA inhalation resulted in an excess of 1.7 µg/g wet of Ni and 0.5 µg/g wet of V. Therefore, the amount of ROFA deposited based on Ni and V is


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TABLE 5 Analyses of Guinea Pig Lung for Vanadium and Nickel Content following Residual Oil Fly Ash (19–20 mg/m3, 2 h) Inhalation
 



    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The first and primary goal of the present study was to investigate the effect of C and GSH depletion in guinea pigs on lung and nasal injury resulting from inhalation of residual oil fly ash. Lung lavage fluid and cell concentrations of GSH and C were lowered by pharmacologic and dietary treatments, then guinea pigs were exposed to ROFA; lung lavage fluids were collected immediately or 24 h later (while guinea pigs were being maintained in the depleted state). The depletion regimens appeared to be effective in lowering the concentrations of C and GSH (by 50–90%) in cells and supernatants of lung lavage. ROFA exposure was found to exert toxic effects only in the animals that had been depleted of both C and GSH. These effects included increased BAL fluid concentrations of protein and LDH, increased BAL neutrophils, and decreased BAL macrophages. Thus, our original hypothesis, that antioxidant depletion might lessen the toxicity of inhaled ROFA, was disproved. Instead, the exact opposite result was obtained.

The fact that toxicity indicators were not altered in control or in singly antioxidant-depleted guinea pigs was probably the result of the relatively low exposure to ROFA. Exposure of rats to a similar air concentration of ROFA, but for 6 h/day for 3 days (rather than a single 2-h exposure used here) resulted in a significant increase in BAL protein and LDH. Even so, ROFA deposition on a per-lung basis in the present study was probably still much higher than a dose considered high for ambient air-exposed humans. The amount of ROFA deposited was calculated from the Ni content of the guinea pig lung and the concentration of Ni in ROFA. The fact that ROFA deposition calculated from the V content of the lung was lower (12.5 µg/g lung) than that calculated from the Ni content (46 µg/lung) can be explained by the fact that V is cleared more rapidly from the lung (18 min half-life; Rhoads and Sanders, 1985) than Ni (30 days; English et al., 1981). A crude comparison of guinea pig ROFA deposition with that of a human can be made as follows. A human (nonexercising) might breathe ~10 m3 of air in 24 h, and if exposed to 150 µg/m3 of PM (the National Ambient Air Standard for PM of 10 micron diameter or smaller; U.S. EPA, 1996), with a lung (airways plus alveoli) deposition of 20% of inhaled (Rudolf et al., 1990Go), about 300 µg of ROFA would be deposited in lungs weighing approximately 300 times more than the guinea pig lung (resulting in 1 µg ROFA/g wet lung).

The second goal of the present study was to determine whether ROFA inhalation alters antioxidant concentrations in nasal and lung lining fluids. ROFA exposure decreased C concentrations in both fluids (Table 3Go). In cases in which deficiency was already established by diet or drug treatment, no further C or GSH depletion by ROFA was detected. NL C was depleted more than BAL C, and the decrease was still significant in NL 24 h postexposure. BAL C was decreased by ROFA at 0 h, but rebounded to higher than normal levels at 24 h. GSH was never decreased by ROFA, but was increased in BAL at 24 h. This increase was not influenced by C status (although it was prevented by GSH depletion). Cellular concentrations of antioxidants tended to be less affected by antioxidant depletion regimens, and they were not affected as much by ROFA exposure. For example, BAL cell C levels were not lowered by ROFA exposure. The time course of changes in cells also appeared to be different than that of the BAL supernatant. BAL cell GSH was increased at 0 h post-ROFA exposure, whereas BAL supernatant GSH levels were not elevated until 24 h (Table 3Go). Also evident from Table 3Go is that the exposure regimen itself elevated both C and GSH and that some compensatory elevations occurred in one antioxidant when the other was depleted. Previous studies have suggested that C and GSH can spare one another in induction of scurvy (Martensson et al., 1993Go) and in ozone-induced injury (Kodavanti et al., 1995Go). To our knowledge, the present study is the first report of the influence of a double deficiency in C and GSH on a toxic response.

Studies are underway to determine the significance for the host of BAL and NL antioxidant changes induced by inhaled particles. Zielinski et al., (1999) suggested that the loss of antioxidants induced by particles might increase the chances for subsequent epithelial cell injury. The present results show no evidence that the loss of antioxidants per se induces injury. In fact, whenever we were able to observe a ROFA-induced C depletion acutely, or a GSH and C replenishment at 24 h, the guinea pigs were spared of injury. We suggest that the ability to regenerate both C and GSH during the 24-h postexposure period somehow prevents the toxic response. This idea is supported by recent findings that ROFA-induced injury in spontaneously hypertensive rats is greater than in normal rats, and the former rats also have a decreased ability to regenerate C and GSH in BAL fluid (Kodavanti et al., 2000Go). A further study in which BSO alone is used to prevent regeneration of GSH after ROFA exposure (in the absence of GSH depletion) might elucidate this possibility.

Other possible mechanisms by which C and GSH could prevent ROFA-induced injury might include stimulation of acute oxidation, which in turn stimulates cellular repair; inhibition of the initial injury by scavenging of phagocyte-derived oxidants released extracellularly; inhibition of metal binding to oxidation targets; or stimulation of redistribution or sequestration of metals. A good discussion of these possibilities is beyond the scope of this paper. In a separate study, we show that human lung epithelial lining fluid (as well as a synthetic fluid) is rapidly auto-oxidized by ROFA in an in vitro system that measures oxygen-18 gas incorporation as a measure of oxidation (Sun et al., 2001Go). This autoxidation is stimulated by physiological concentrations of C and, to a lesser extent, GSH. We showed previously that ROFA, as well as individual metals found in ROFA, stimulate rat alveolar macrophages to generate oxidants detectable by chemiluminescence (Ghio et al., 1997Go). It has not yet been possible to detect the same oxidative reactions in vivo that have been demonstrated in vitro.

Our present findings are in general agreement with a series of recent studies involving in vivo iron loading and in vitro supplementation of cells or blood plasma with FE and C. For example, liver microsomes from guinea pigs fed both Fe and C were less susceptible to autoxidation than C-depleted guinea pigs (Collis et al., 1997Go). Also, iron-overloaded guinea pigs that were supplemented with C had lower F2-isoprostane levels (a sensitive indicator of lipid peroxidation) in the plasma and liver than nonsupplemented guinea pigs (Chen et al., 2000Go). In vitro studies have shown that C supplementation of 3T3 fibroblasts ameliorates the toxicity of ferrous ions (Collis et al., 1996Go), and that blood plasma supplemented with C is less susceptible to LDL oxidation by iron (Berger et al., 1997Go).

Previous studies of ROFA toxicity suggest an oxidative mediation of some of the toxic responses. Intratracheal instillations of ROFA in rats caused BAL cellular changes that were partially inhibitable by injection of the synthetic antioxidant dimethylthiourea (Dye et al., 1997Go). Exposure of guinea pig tracheal epithelial cells to ROFA resulted in LDH release by and induction of mucin genes. GSH depletion enhanced these toxic processes, and dimethylthiourea inhibited them. Further work will be required to define the role of antioxidants in the toxicity of fly ash metals. Goals should include improving methods for measuring oxidation in vivo; elucidating mechanisms responsible for alterations in in vivo indicators of toxicity; determining effects of metal mixtures in a larger array of antioxidant concentrations; understanding intracellular signaling induced by oxidation; and understanding the mechanisms of in vivo regeneration of C and GSH at target sites.

The third goal of the study was to obtain general information about the effect of a combined deficiency of C and GSH. Some effects were noted in the absence of ROFA exposure. For example, BAL eosinophil counts (although not affected by ROFA) were increased at 24 h by both C- and GSH-depletion regimens. The GSH-depletion regimen was also associated with increased total cell counts (including both macrophages and eosinophils) at 24 h post-ROFA. We also found that the GSH-depletion regimen lowered C concentrations in BAL at 0 h and lowered rectal body temperature. We are not aware of previous reports of either of the above side-effects of the GSH-depletion regimen we used. The dose of diethylmaleate we used (1.2 mmol/kg) was 1/15 of the LD50 (18 mmol/kg ip in rats). We used the latter agent because we determined previously that BSO alone, under conditions used in the present study, did not lower lung GSH in guinea pigs (Slade et al., 1989Go).

Based on the present data, it is clear that excess C would not be a risk factor for exposure to ROFA. Conversely, it is likely that depletion of both C and GSH might be a risk factor for exposure to ROFA or similar metal-containing PM. A recent study showed that lymphocytes from elderly people have 10–20% lower concentrations of C and GSH in subjects over 60 years of age than younger subjects (Lenton et al., 2000Go). A seasonal variation was also noted in all age groups, with concentrations of both antioxidants as much as 38% higher in summer than winter. The fact that normal humans have much lower BAL concentrations of both C and GSH (90% lower, Slade et al., 1993) compared with rats and guinea pigs also suggests that rodent inhalation toxicology studies on ROFA probably underestimates toxicity in humans.

In summary, dietary depletion of C and /or GSH in guinea pigs produced decreases in C and GSH basal concentrations in NL and BAL fluids and cells. Combined deficiency of both C and GSH made guinea pigs susceptible to injurious effects of ROFA inhalation (measured by increases in lung lavage fluid protein, LDH, neutrophil counts, and decreases in macrophage counts). C was depleted in NL and BAL by ROFA, and both C and GSH were regenerated in excess amounts in BAL fluid during the 24-h period following exposure to ROFA in normal guinea pigs. Therefore, it appears that both C and GSH are important in protection from ROFA toxicity, and that the basal concentrations of both antioxidants or their ability to be regenerated following ROFA exposure are responsible for their protective effects.


    ACKNOWLEDGMENTS
 
The authors graciously thank Drs. Urmila P. Kodavanti and Daniel Morgan for their critical review and helpful suggestions on this manuscript, Kay M. Crissman for water-soluble antioxidant analyses, Judy H. Richards for biochemical analyses using the COBAS-FARA II automatic analyzer, and Ralph Slade, Linda P Harris, and John L. McKee for their assistance with cell preparation.


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

1 To whom correspondence should be addressed. Fax: (919)541-0026. E-mail: hatch.gary{at}epa.gov. Back


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