Rapid increases in the steady-state concentration of reactive oxygen species in the lungs and heart after particulate air pollution inhalation. (Articles).
Ambient air particles are chemically complex and include minerals, organics, and biologic air pollutants. Epidemiologic studies have shown that increased levels of ambient airborne particulate matter (PM) are associated with increased cardiopulmonary morbidity and mortality (reviewed by Kaiser 2000). Particulate air pollution shares some physicochemical properties with mineral dusts known to act through oxidant mechanisms, such as silica and asbestos (reviewed by Churg et al. 1997; Mossman 2000). Known source constituent particles such as oil fly ash, coal fly ash, and diesel exhaust, extensively used as surrogates of PM, are also effective pro-oxidants in vitro as well as in vivo (Baeza-Squiban et al. 1999; Kadiiska et al. 1997; Nel et al. 2001; Stringer and Kobzik 1998), suggesting that PM toxicity may be due to increased generation of reactive oxygen species (ROSs) in target cells.
In the last few years, the study of the intrinsic toxicity of "real-world" ambient air particles has notably expanded due to the development of the technology to collect, sort, and concentrate PM from urban air samples without altering their physicochemical properties (Sioutas et al. 1995). Some of the epidemiologic findings on the health effects of PM, including inflammation and toxicity, have been successfully reproduced in the laboratory in humans (Ghio et al. 2001), dogs (Clarke et al. 2000b), and rats (Clarke et al. 2000a).
In vitro studies have also showed a variety of biologic responses to concentrated ambient particles (CAPs), including redox regulation and proliferation (Jimenez et al. 2000; Timblin et al. 1998), increased production of proinflammatory cytokines (Imrich et al. 1999; Monn and Becker 1999), increased oxidation of redox-sensitive fluorescent dyes (Baeza-Squiban et al. 1999; Goldsmith et al. 1998; Prahalad et al. 1999; Shukla et al. 2000), and transcriptional activation of redox-sensitive genes (Shukla et al. 2000). Although the in vitro findings support the hypothesis that ROSs are mediators of PM biologic effects, we have no direct evidence to date of a particle-driven increased production of oxidants in vivo.
In this study, we used inhalation exposure of rats to CAPs aerosols, combined with measurements of in situ chemiluminescence (CL), to evaluate the ability of CAPs to increase ROS concentrations in intact animals in real time and in a noninvasive manner. CL is a low-intensity emission in the visible range mainly due to the decay of excited states of molecular oxygen (singlet oxygen and excited carbonyls; Boveris et al. 1980; Cadenas and Sies 1984), which are formed during the termination steps of the chain reaction of lipid peroxidation (Halliwell and Gutteridge 1990). The spontaneous CL of the organs in situ correlates with the square of the intracellular concentration of [H.sub.2][O.sub.2] and with the development of oxidative damage (Boveris and Cadenas 1997; Gonzalez-Flecha et al. 1993). Measurements of low-level CL have been used to assess the concentration of oxidants in several models of toxicity to the lung, heart, and liver. Acute administration of paraquat, in doses known to cause extensive lung damage (30 and 60 mg/kg body weight), produced > 100% increases in lung CL (Turrens et al. 1988). Perfusion of isolated rat lungs with tert-butyl hydroperoxide or activated polymorphonuclear leukocytes resulted in 200-400% increases in CL and were associated with significant edema and accumulation of thiobarbituric acid-reactive substances (TBARSs) (Barnard et al. 1993). In the mouse heart, measurements of CL have been used to study the differential toxicity of the antitumor drugs adriamycin and mitoxantrone. Acute administration of adriamycin to mice resulted in 10-fold increases in spontaneous heart CL and 80% increases in TBARS accumulation. In contrast, administration of mitoxantrone, a functional analog with lower toxicity, did not affect the production of free radicals or the accumulation of oxidized lipids in the heart (Lores Arnaiz and Llesuy 1993).
In a previous study we used this technique to quantify the increases in the steady-state concentrations of oxidants associated with the development of oxygen tolerance in a model of adaptation to mild hyperoxia (Evelson and Gonzalez-Flecha 2000). We show here that inhalation of ambient air particles, but not control inert particles, rapidly increases the steady-state concentrations of oxidants in the lung and heart but not in the liver. The oxidative stress imposed by CAPs is associated with the metal content in particles in a tissue-specific manner and leads to mild increases in lung and heart edema as well as in serum levels of lactate dehydrogenase (LDH). Animals breathing CAPs for 5 hr also show an increase in the activity of several antioxidant enzymes in both heart and lung.
Materials and Methods
CAPs. We used the Harvard Ambient Particle Concentrator (HAPC) to concentrate ambient air particles for subsequent aerosol exposure of animals (Sioutas et al. 1995). The principle of virtual impaction was used to concentrate ambient particles in the size range of 0.1-2.5 [micro]m (fine particles; concentration factor, 26 [+ or -] 4; Sioutas et al. 1995). CAPs remained in suspension without physical or chemical alteration for inhalation exposures or for collection onto filters for mass and composition analysis. During the operation of the HAPC, we continuously monitored mass concentrations (gravimetrically determined) and the size of the particles (using a microorifice impactor) (Godleski et al. 2000). We determined trace metal concentrations using X-ray fluorescence (Chester LabNet, Tigrad, OR, USA).
Exposure to CAPs. We used pathogen-free male Sprague-Dawley rats (Taconic Farms, Germantown, NY, USA) weighing 250-300 g. Animals were fed a conventional laboratory diet and water ad libitum. We exposed rats to CAPs aerosols (CAPs group) or filtered air (control group) in the chamber of the HAPC (Clarke et al. 2000a). The animals were awake and unrestricted during the exposures, and we exposed and tested the CAPs and control groups simultaneously. We carried out each exposure with groups of six animals: three were exposed to CAPs aerosols and three to filtered air (sham controls). At 1, 3, and 5 hr, we removed two animals (one exposed to CAPs and one sham control) from the chamber to be assessed for oxidative stress, tissue damage, and antioxidant enzymes as described below. We conducted each experiment with groups of two animals. The temperature in the room and chamber was 25[degrees]C.
Exposure to carbon black and ROFA. We carried out exposures to carbon black and residual oil fly ash (ROFA) in a 40 x 25 x 60 cm chamber. We used a Wright dust feeder (model MK-II; L. Adams Ltd., London, UK) to generate carbon black and ROFA aerosols. Carbon black (catalog no. C198) was purchased from Fisher Scientific (Pittsburgh, PA, USA). We analyzed the elemental composition of carbon black by X rays using a LEO 1450 VP scanning electron microscope with an Oxford Si detector (Leo Microscopy, Inc., Thornwood, NY, USA). Carbon black particles consisted of 85.9 [+ or -] 0.2% carbon, 13.0 [+ or -] 0.2% oxygen, and 1.17 [+ or -] 0.02% sulfur. We detected no transition metals. We obtained fly ash from a Boston, Massachusetts, area oil-fired power plant (Killingsworth et al. 1997). The metal content of ROFA was as reported by Killingsworth et al. (1997). We packed ROFA or carbon black particles into the dust feeder and flushed it with a stream of air at 14 L/min (6 pounds per square inch gauge). We passed the air stream containing the aerosols through a size-selective impactor (to eliminate particles > 2.5 lam) and then fed it into the exposure chamber isokinetically. We determined particle concentration from the mass change on the filter and the total volume of air sampled. We also monitored the particle concentration during exposures using a real-time aerosol monitor (model RAS-1; MIE Inc., Bedford, MA, USA).
Exposure to filtered air. For the experiments in which rats were exposed for 3 days to either room air or filtered air, long-term continuous exposures to filtered air took place in a 40 x 40 x 60 cm chamber. We used a Millipore 0.2 [micro]m filter to retain PM, and filtered air was humidified with sterile water before delivery to the chamber at a flow rate of 12 L/min. The temperature in the room and chamber was maintained at 25[degrees]C.
Organ CL. We measured CL of the lung, heart, and liver in situ as previously described (Evelson and Gonzalez-Flecha 2000) using a Thorn EMI CT1 single-photon counting apparatus with an EMI 9816B photomultiplier (Electron Tubes, Inc., Rockaway, NJ, USA) cooled at -20[degrees]C. Rats were anesthetized (sodium pentobarbital, 50 mg/kg body weight) and connected to an animal ventilator (5 mL/breath, 60 breaths/rain; Harvard Apparatus, Cambridge, MA, USA). Once we intubated and ventilated the animal, we opened the chest and placed the animal in the measurement compartment. We carried out the surgical procedure and measurements in < 10 min, allowing analysis to begin within 15 min of CAPs exposure. We kept rats at ~37[degrees]C using isothermal pads (Braintree Scientific, Braintree, MA, USA). The emission data is expressed as counts per second per unit of tissue surface (cps/[cm.sup.2]). We placed a high-pass cutoff filter (Wratten no. 25; Eastman Kodak, Rochester, NY, USA), which allows wavelengths > 600 nm, in the optical path to avoid hemoglobin interference. Photon counting decreased only by 15-20%, thus indicating that 80-85% of the emitted light could be regarded as singlet oxygen emission (singlet oxygen dimol emission, 634 and 703 nm; Cadenas and Sies 1984).
Tissue preparation. After measuring CL, we removed the animals from the ventilator; we then rapidly removed the lungs, liver, and heart, and froze them in a dry ice bath. We took separate samples from each tissue and time point to determine water content or enzymatic activities. We also withdrew blood samples from the inferior vena cava to determine serum markers of tissue damage [LDH and creatine phosphokinase (CPK) activities]. We homogenized samples for the determination of enzymatic activities in 5 volumes of 120 mM KCl, 30 mM phosphate buffer (pH 7.2) with added protein inhibitors (1 [micro]g/mL leupeptin, 1 [micro]g/mL aprotinin, 10 [micro]g/mL soybean trypsin inhibitor, 1 [micro]g/mL pepstatin, and 0.5 mM phenylmethyl sulfonyl fluoride) at 0-4[degrees]C. We centrifuged the suspensions at 600 x g for 10 min at 0-4[degrees]C to remove nuclei and cell debris. We discarded the pellets and used the supernatants as homogenates.
Enzymatic measurements. We measured fumarase activity by following the increase in absorbance at 240 nth at 25[degrees]C in a reaction mixture containing 30 mM phosphate (pH 7.4), 0.1 mM EDTA, and 5 mM L-malate (Racker 1950; Sigma Chemical Co., St. Louis, MO, USA). We determined total superoxide dismutase (SOD) activity from the rate of inhibition of the oxidation of 20 [micro]M ferrocytochrome c at 550 nm in a reaction mixture consisting of 50 [micro]M phosphate buffer (pH 7.8), 50 pM xanthine, and 5 mU xanthine oxidase (McCord and Fridovich 1969). We measured MnSOD activity after inhibition of the Cu/Zn isoenzyme by addition of 1 mM KCN (Beauchamp and Fridovich 1973). We determined catalase activity by measuring the decrease in absorption of [H.sub.2][O.sub.2] at 240 nm in a reaction medium containing 2 mM [H.sub.2][O.sub.2] (Nelson and Kiesow 1972). We determined hemoglobin on rat lung homogenates using a standard kit (Sigma Chemical Co., St. Louis, MO, USA) and quantified it by comparison with standard hemoglobin solutions. SOD activity attributable to hemoglobin represented 1-5% of the total SOD activity in both lung and heart. We measured protein concentration in homogenates by the method of Lowry et al. (1951) using bovine serum albumin as standard. We carried out measurements in a Perkin Elmer Lambda 40 spectrophotometer (Perkin-Elmer, Norwalk, CT, USA).
Serum markers of tissue damage. We measured LDH and CPK activity and hemoglobin content in serum samples spectrophotometrically using standard kits (Sigma Chemical Co.).
Water content. We weighed lung and heart samples (~100 mg) and then dried them in a conventional oven (~80[degrees]C). We reweighed tissues after 24 hr to obtain the wet/dry ratios.
Statistics. Values in tables and figures are mean [+ or -] SEM. We analyzed data statistically by factorial analysis of variance followed by Fisher's test for comparison of means. For elemental composition correlation analyses, we fitted separate linear regression models using actual elemental concentration univariately as predictors. We performed all statistical analyses using Statview software (Abacus Concepts, Inc., Berkeley, CA, USA) for Macintosh. We carried out graphical diagnostics of model adequacy and outlier detection using the S-Plus statistical package (Mathsoft, Inc., Seattle, WA, USA) (Venables and Ripley 1994).
Animal care. Animals were handled humanely in the performance of this project to minimize the use of animals and to prevent animal distress. All protocols of exposure and other procedures used in this study were approved by the Harvard Animal Use Committee. The Harvard School of Public Health is accredited by the American Association for the Accreditation of Laboratory Animal Care, meets National Institutes of Health standards as set forth in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources 1996), and accepts as mandatory the National Institutes of Health's Principles for the Use of Animals (NIH 2000).
CAPs increases the steady-state concentration of oxidants in the lung and heart. Inhalation exposure to CAPs increases the steady-state concentration of oxidants in the rat lung and heart. In vitro studies suggest that biologic effects of particulate air pollution are initiated by an increased generation of ROS in cells exposed to particles. To determine whether PM affects ROS production in intact animals, we exposed adult Sprague-Dawley rats to aerosols of CAPs and monitored the steady-state concentration of oxidants in the lung, heart, and liver by measuring their spontaneous in situ CL. We chose to study the lung and heart because they are the major targets of particulate air pollution (Kaiser 2000). The central role of the liver in the detoxification of a wide range of xenobiotics suggests that this organ may also be a target for the soluble fractions of inhaled particles.
Figure 1 shows the mean values of in situ CL in the lung, heart, and liver of rats exposed to CAPs or filtered air for 1-5 hr. Our data show a significant increase in lung and heart CL at 5 hr of exposure. The time courses of increase in CL in the lung and heart follow slightly different patterns. Lung CL increased linearly with the time of exposure (Figure 1A). On days of high pollution (CAPs concentration > 500 [micro]g/[m.sup.3]), differences between the CAPs and filtered air groups were apparent after only 1 hr of exposure (data included in Figure 1A). In contrast, heart CL showed a lag phase of about 1 hr before any increase could be detected (Figure 1B). The increases in lung and heart CL were specifically due to inhaled CAPs because they were absent in the control animals breathing filtered air under the same experimental conditions (Figure 1). Liver CL was unchanged throughout the 5 hr of exposure to CAPs (Figure 1C).
[FIGURE 1 OMITTED]
To determine if the oxidant effect of CAPs in lung and heart tissue depended on particle composition, we tested model environmental particles of different composition for their pro-oxidant effects in lung and heart (Table 1). ROFA is composed of fugitive oil combustion particles, which contribute to PM in urban air and have been shown to cause pulmonary injury and inflammation (Kodavanti et al. 2001; Madden et al. 1999; Nadadur et al. 2000). The large batch of ROFA particles available allows a constant composition of particles during experimental exposures. ROFA is rich in transition metals, specifically vanadium, Fe, and nickel (Killingsworth et al. 1997). Carbon black fine particles resemble the carbonaceous core of PM, but because of their synthetic origin, they do not carry significant levels of adsorbed metals or organic compounds. Consistent with this lack of active components, fine carbon black particles are mostly inert in in vivo systems; therefore, we used them as negative control particles (Killingsworth et al. 1997; Murphy et al. 1998).
Table 1 shows that exposure to inert carbon black particles does not exert oxidant effects on the heart or lung. In contrast, ROFA aerosols produced a strong increase in lung and heart CL after exposures as short as 30 min (Table 1). These results strongly suggest that the oxidant effect of environmental particles (CAPs and ROFA) is due to specific components not present in the chemically inert carbon black particles.
To further test this thesis, we took advantage of the day-to-day variations in CAPs composition, which provided a set of samples with a range of metal concentrations sufficient for statistical analyses (Table 2). Using univariate regression analyses, we identified several components with significant associations to increased CL in the lung or heart (Table 3). Because of the strong effect of CAPs inhalation on both lung and heart CL, many elements show positive correlations with the CL levels (Table 3). However, we found the stronger and more significant associations for Mn, Fe, Cu, and Zn in the lung and for total mass, Al, Si, Ti, and Fe in the heart (Table 3).
The effect of CAPs on the steady-state levels of oxidants could be due to reversible or irreversible interactions of the particles with cellular components. To determine what type of interaction was responsible for the observed increases in CL in this model, we used two complementary approaches. In one approach we simulated transient increases in particulate air pollution by exposing rats to CAPs for 5 hr and then allowing them to recover in ambient air (room air) for 24 hr. In the other approach, we exposed rats to particle-free (filtered) air for 3 days, which is known to decrease the concentrations of oxidants in the lung and heart (Evelson and Gonzalez-Flecha 2000), and then allowed them to recover in room air for up to 8 hr. Table 4 summarizes the effects of both treatments on the steady-state concentrations of oxidants in the lung. Rats breathing CAPs for 5 hr showed a significant increase in their in situ lung CL. However, CAPs-initiated oxidative stress is not detectable in rats allowed to recover in room air after the simulated "peak" in particulate air pollution (Table 4). Similarly, the second approach showed that decreases in ambient particle levels also affect ROS production in the lung, and this effect is also reversible. Rats breathing particle-free filtered air for 3 days had significantly lower levels of oxidants in their lungs (Table 4). As in the model of acute exposure to increased pollution, the effect of chronic "depletion" in PM is reversed shortly after reexposure to room air.
CAPs-induced oxidative stress is associated with mild damage to the lung and heart. To assay for lung and heart damage in this model, we measured water content (edema) and serum levels of LDH and CPK in rats exposed to CAPs for 5 hr. We measured these markers in blood and tissue samples collected from the same rats assayed for CL (Figure 1). The water content of both lung and heart increased significantly upon exposure to CAPs but not to filtered air (Table 5). The wet/dry ratio in both tissues showed significant increases as a function of the length of exposure and with respect to the control (filtered air) animals for exposure times longer than 3 hr (Table 5). In agreement with the lack of oxidative unbalances in the liver, the wet/dry ratio in this tissue remained unchanged throughout the exposure (filtered air sham, 3.5 [+ or -] 0.1; CAPs 3 hr, 3.5 [+ or -] 0.1; CAPs 5 hr, 3.3 [+ or -] 0.1). Because we collected these samples immediately after the end of the exposure, the observed increase in water content indicates an almost immediate interaction (and toxicity) of environmental particles with lung and heart cells. To evaluate longer-term responses, we also studied rats exposed to CAPs or filtered air for 5 hr and tested for lung and heart edema and serum markers of tissue damage 24 hr after the end of the exposure (Tables 5 and 6). Interestingly, the wet/dry ratio in rats breathing room air for 24 hr after 5 hr of exposure to CAPs returned to control values in the lung but not in the heart (Table 5). These results indicate that the lung can readily compensate for transient increases in the levels of ambient air particles. In contrast, the effects of CAPs on the heart tissue are more pronounced and longer lasting.
Rats breathing CAPs also showed increases in the serum levels of LDH and CPK, as a function of the length of exposure and compared with filtered air controls (Table 6). LDH activity increased approximately 2-fold in rats exposed to CAPs for 5 hr and returned to control values 24 hr after the end of the exposure. CPK activity also showed a significant increase in the serum activity as a function of the time of exposure to CAPs but not to filtered air (Table 6). However, because of a slight increase in the CPK values of the control group, the differences between CAPs and filtered air control groups for the same times of exposure did not reach statistical significance (Table 6). As in the case of LDH release, CPK activity returned to control levels in animals tested 24 hr after the end of a 5-hr exposure to CAPs. For both enzymes, the increase in serum activity is mild and compatible with reversible tissue damage.
Short-term exposure to CAPs up-regulates antioxidant activities. In addition to their effect on the intracellular production of ROS, some xenobiotics can also affect antioxidant defense systems (Lissi et al. 1991). To test whether this was the case for CAPs, we measured the activity of SOD and catalase (the main detoxifying systems for superoxide and hydrogen peroxide, respectively) in tissue samples collected from rats exposed to CAPs or filtered air for 5 hr (Figure 1). Because of the short exposure and the relatively low toxicity of CAPs, we expected to see no change in these antioxidants. However, our data showed an increase in SOD and catalase activities in both lung and heart (Table 7). The pattern of increase in these activities was tissue specific. In the lung, we found the higher level of induction (70%) for MnSOD, the mitochondrial isoform of SOD (Table 7). Cu/ZnSOD, the cytosolic isoform, was unchanged, and catalase was increased by 30%. In the heart, in contrast, Cu/ZnSOD showed the highest level of induction (100%), MnSOD was increased by 40%, and catalase by 20% (p > 0.05).
To confirm that the increases in SOD and catalase activities were due to specific regulatory effects and not to a global effect on proteins, we measured the activity of fumarase, an essential tricarboxylic acid cycle enzyme resistant to oxidants (Evelson and Gonzalez-Flecha 2000). Fumarase activity was unchanged in rats exposed to CAPs compared with their filtered air controls.
Epidemiologic studies have shown that exposure to airborne PM is associated with increased cardiopulmonary morbidity and mortality. The mechanisms by which particulate air pollution causes cardiopulmonary effects at the cellular level are poorly understood. The ability of PM to increase the intracellular production of ROS, although assumed to be essential for their biologic effects, has never been tested in vivo. In the present study, we have taken advantage of two unique tools, the HAPC and the measurements of organ CL, to begin to investigate the postulate that particle toxicity operates through oxidant-dependent mechanisms derived from specific particle components. We found that inhalation of CAPs increases by 2-fold the steady-state concentration of ROS in the rat lung and heart. Organ CL measures the steady-state concentration of singlet oxygen (102), a by-product of lipoper-oxidation, in intact organs. In this way, organ CL provides an accurate measure of the redox status of the tissue. The spontaneous CL of the organs in situ has been successfully used to assess oxidant stress in several models of toxicity to the lung, heart, and liver. The magnitude of increase in lung and heart CL observed in rats breathing CAPs for 5 hr is similar to that previously reported by us in rats exposed to 85% [O.sub.2] for 3-5 days, a treatment associated with mild and transient lung and heart injury (Evelson and Gonzalez-Flecha 2000), and by Turrens et al. (1988) in rats treated with sublethal doses of paraquat (30 mg/kg, intraperitoneal).
In our model, in situ CL of the lung increases shortly after exposure to CAPs (Figure 1) and returns to control values a few hours after removal of the animals from the HAPC (Table 4). The rapid increase in the lung concentrations of ROSs upon exposure to CAPs indicates an almost immediate effect of particles, or particle components, on the intracellular sources of free radicals. Furthermore, the transient nature of these increases points to a reversible interaction of particle components with cellular targets. Both observations would be compatible with Fenton-type reactions catalyzed by transition metals, redox-cycling processes, or biochemical changes triggered by noncovalent binding to membrane receptors. Single-component regression analyses showed a strong association of the oxidant effect of CAPs aerosols generated on different days and their content of transition metals, specifically to the CAPs content of Mn, Zn, Fe, and Cu (Table 3). The notable lack of association with the total mass of particles strongly supports a cause-effect relationship between the presence of these metals and the oxidant capability of CAPs aerosols, as opposed to a nonspecific effect caused by the physical interaction of foreign particles with lung cells. The association of the CAPs oxidant effect with their content of redox-active metals also supports the idea of an increased occurrence of Fenton-type reactions in the lung of CAPs-exposed animals.
These results agree with previous reports showing associations of different biologic readouts with the levels of transition metals in CAPs. Intratracheal instillation of ROFA particles with high Mn content induced bronchoalveolar lavage (BAL) eosinophilia in vivo (Jiang et al. 2001). Inhalation of soluble Fe, V, and Ni sulfates showed substantial cardiopulmonary toxicity in rats with acute or subacute ozone-induced pulmonary inflammation (Watkinson et al. 2001). Addition of surface iron converts nonreactive titanium dioxide particles into fibrinogenic particles (Dai et al. 2001). Finally, in a model similar to ours but with exposure time of 6 hr/day on 3 consecutive days, CAPs inhalation in rats elicited acute lung inflammation and the intensity of the inflammation was associated with the levels of metals (Saldiva et al. 2001).
The effect of CAPs on the ROS concentrations in the heart seems to operate through a different series of mechanisms. In contrast to lung CL, heart CL showed a 1-hr lag phase (Figure 1B) that may reflect the time required for the lung cells to signal the heart of the presence of an oxidant insult. The oxidant effect of CAPs on the heart is associated with CAPs components different from those associated with the development of oxidative stress in the lung. Heart CL strongly correlated with the CAPs content of Al, Si, Ti, and Fe, as well as with the total mass. The common association of lung and heart CL with Fe content suggests that at least some of the effects may be due to direct mechanisms, probably Fenton-type reactions. However, indirect mechanisms are also suggested by the associations of heart CL with nonredox active components such as Si and Ti and with the total CAPs mass. Reports by Clarke et al. (2000a, 2000b) also show association of increased pulmonary neutrophil percentages with the content of Al and Si in CAPs.
We also found that 5-hr inhalation exposure to CAPs causes significant tissue edema (Table 5) and increased release of LDH (Table 6). These results agree with the increased levels of BAL neutrophils and circulating lymphocytes reported in rats exposed to CAPs for 3 days (Clarke et al. 2000a). The slight increases in water content ([less than or equal to] 5% for both tissues; Table 5) and release of LDH ([less than or equal to] 80%; Table 6) and CPK ([less than or equal to] 50%; Table 6) reported here for the short-term exposures tested are compatible with mild, reversible damage. As for the increases in lung and heart CL, the magnitude of increase in the lung and heart water content was similar to that reported in rats breathing 85% [O.sub.2] for 3 days (Evelson and Gonzalez-Flecha 2000), a treatment associated with significant, although not lethal, injury to the lung and heart. In contrast, the increases in LDH and CPK values observed in animals breathing CAPs, although indicative of an increased permeability in cellular membranes, are below the levels associated to massive morphologic changes (Gonzalez-Flecha et al. 1993).
One of the most striking findings of this study is that particulate air pollution has the ability to up-regulate antioxidant enzymes. We found that 5-hr exposures to 100-500 [micro]g/[m.sup.3] CAPs increased SOD and catalase activities in a tissue-specific manner (Table 7). MnSOD induction was more marked in the lung than in the heart (70% vs. 40%), whereas increases in Cu/ZnSOD are observable only in the heart (100%). On the other hand, catalase.was increased by 30% in the lung and showed a trend of increase that did not reach statistical significance in the heart. These patterns of antioxidant enzyme induction agree with results from gene array studies of the responses to inhaled CAPs in the rat lung (Godleski JJ. Personal communication). In these experiments, rats exposed to CAPs aerosols for 6 hr/day on 3 consecutive days showed increased lung mRNA levels of MnSOD and catalase. In contrast, expression of Cu/ZnSOD was slightly decreased after exposure. Taken together, these results suggest transcriptional regulation of antioxidant enzymes by short-term exposures to CAPs. Up-regulation of antioxidant enzymes has been described in other models of oxidant inhalation. Rats breathing 85% oxygen for > 5 days develop resistance to 100% oxygen, and this increased tolerance is associated with higher levels of MnSOD and Cu/ZnSOD in the lung (Clerch and Massaro 1993; Crapo and McCord 1976). Long-term exposure to ozone has also been reported to cause site-specific increases in the activities of antioxidant enzymes (Plopper et al. 1994).
There is abundant data that MnSOD can be induced by ROS (reviewed by Crawdford 1999) and proinflammatory cytokines in vitro (reviewed by Valentine and Nick 1999). Cu/ZnSOD, although found unresponsive to ROS levels and many cytokines, can be up-regulated by interleukin-1 (Tannahill et al. 1997), during cell differentiation (Valentine and Nick 1999), and in response to oxygen in very premature newborn baboons (Morton et al. 1999). Finally, catalase regulation by hydrogen peroxide has been documented in several systems (Csonka et al. 2000; Rohrdanz et al. 2001). CAPs exposure increases ROS concentrations (Figure 1, Table 4) as well as the levels of proinflammatory cytokines (Calderon-Garciduenas et al. 2001; Shukla et al. 2000). Therefore, the observed increases in activity of SODs and catalase could be due to transcriptional activation mediated by these factors.
In summary, our data show for the first time that short-term inhalation exposure to increased concentrations of particulate air pollution promotes oxidative stress and mild damage to the lungs and heart in vivo. The observed up-regulation of antioxidant defenses and the reversibility of the CAPs-mediated oxidative stress and toxicity strongly suggest that the lung and heart can readily adapt to rapid increases in the intracellular levels of oxidants. Further experimentation is warranted to establish the causal role of oxidants in CAPs toxicity as well as to confirm transcriptional regulation of antioxidant enzymes and establish the mechanism operating this regulation.
Table 1. Lung and heart CL in rats exposed to particles of different composition. CL (cps/[cm.sup.2]) Treatment (concentration/time) Lung Filtered air (0 [micro]g/[m.sup.3], 5 hr) 24 [+ or -] 1 ROFA (1.7 mg/[m.sup.3], 30 min) 90 [+ or -] 10 * Carbon black (170 [micro]g/[m.sup.3], 5 hr) 22 [+ or -] 3 Values indicate mean [+ or -] SEM (n = 4-6). CL (cps/[cm.sup.2]) Treatment (concentration/time) Heart Filtered air (0 [micro]g/[m.sup.3], 5 hr) 24 [+ or -] 3 ROFA (1.7 mg/[m.sup.3], 30 min) 50 [+ or -] 3 * Carbon black (170 [micro]g/[m.sup.3], 5 hr) 25 [+ or -] 5 Values indicate mean [+ or -] SEM (n = 4-6). * p < 0.001, compared with filtered air control. Table 2. Elemental composition of CAPs ([micro]g/[m.sup.3]). Total Date mass Al Si S Cl K 7 Jul 2000 130.7 4.986 10.19 3.673 0.209 2.171 9 Aug 2000 957.5 1.513 9.634 110.7 0.000 3.480 10 Aug 2000 334.0 0.534 3.730 29.5 0.051 2.121 11 Aug 2000 352.8 8.938 21.11 17.3 0.005 4.030 17 Aug 2000 99.6 0.901 2.729 4.630 0.230 1.024 23 Aug 2000 328.8 2.208 7.850 32.48 0.148 2.021 8 Sep 2000 229.5 1.437 5.253 20.31 0.156 1.528 15 Sep 2000 219.3 0.000 1.165 27.58 0.434 0.634 29 Nov 2000 226.6 0.449 3.597 29.70 0.390 1.013 11 Dec 2000 465.0 0.417 7.133 76.59 7.015 2.860 18 Dec 2000 121.0 0.159 1.007 5.120 0.124 0.255 8 Feb 2001 255.0 1.319 5.816 14.29 7.210 1.306 22 Feb 2001 219.0 1.678 5.522 10.04 3.549 1.438 Date Ca Ti V Cr Mn Fe 7 Jul 2000 4.690 0.392 0.026 0.013 0.130 5.936 9 Aug 2000 2.720 0.441 0.075 0.028 0.136 7.596 10 Aug 2000 3.408 0.163 0.016 0.011 0.096 3.628 11 Aug 2000 8.962 0.803 0.157 0.038 0.320 11.171 17 Aug 2000 2.721 0.140 0.016 0.013 0.059 3.608 23 Aug 2000 2.111 0.371 0.110 0.027 0.158 6.889 8 Sep 2000 1.891 0.258 0.053 0.028 0.106 4.944 15 Sep 2000 1.451 0.087 0.033 0.028 0.050 2.370 29 Nov 2000 2.299 0.251 0.041 0.014 0.101 6.167 11 Dec 2000 8.987 0.488 0.624 0.024 0.453 13.618 18 Dec 2000 0.550 0.023 0.006 0.012 0.017 0.829 8 Feb 2001 3.644 0.228 0.036 0.014 0.073 4.965 22 Feb 2001 4.640 0.210 0.048 0.015 0.085 4.721 Date Ni Cu Zn As Se Br 7 Jul 2000 0.012 0.079 0.521 0.005 0.000 0.012 9 Aug 2000 0.094 0.156 0.537 0.037 0.111 0.207 10 Aug 2000 0.010 0.112 0.421 0.019 0.020 0.050 11 Aug 2000 0.035 0.232 0.677 0.018 0.000 0.052 17 Aug 2000 0.012 0.084 0.187 0.006 0.002 0.025 23 Aug 2000 0.089 0.150 0.699 0.025 0.022 0.082 8 Sep 2000 0.044 0.145 0.436 0.007 0.082 0.071 15 Sep 2000 0.029 0.056 0.299 0.000 0.022 0.065 29 Nov 2000 0.030 0.188 0.327 0.018 0.013 0.034 11 Dec 2000 0.518 0.346 2.169 0.028 0.062 0.282 18 Dec 2000 0.011 0.021 0.056 0.005 0.000 8.000 8 Feb 2001 0.035 0.113 0.239 0.017 0.002 0.032 22 Feb 2001 0.057 0.093 0.211 0.007 0.001 0.118 Date Cd Ba Pb 7 Jul 2000 0.018 0.376 0.099 9 Aug 2000 0.014 0.709 0.180 10 Aug 2000 0.000 0.469 0.082 11 Aug 2000 0.000 1.222 0.177 17 Aug 2000 0.004 0.466 0.062 23 Aug 2000 0.015 0.545 0.240 8 Sep 2000 0.000 0.468 0.155 15 Sep 2000 0.010 0.369 0.075 29 Nov 2000 0.002 0.504 0.080 11 Dec 2000 0.000 0.992 0.262 18 Dec 2000 0.005 0.293 0.027 8 Feb 2001 0.000 0.416 0.073 22 Feb 2001 0.003 0.436 0.086 Table 3. Statistical parameters for the regression of lung and heart CL and the mass CAPs elemental components. Lung CL Heart CL Element [r.sup.2] p-Value [r.sup.2] p-Value Al 0.14 0.140 0.67 * 0.001 Si 0.31 0.020 0.61 * 0.002 S 0.14 0.140 0.08 0.360 Cl 0.14 0.070 0.08 0.360 K 0.39 0.008 0.30 0.500 Ca 0.41 * 0.007 0.36 0.030 Ti 0.38 0.008 0.59 * 0.002 V 0.02 0.620 0.42 0.020 Cr 0.04 0.440 0.32 0.046 Mn 0.51 * 0.001 0.43 0.010 Fe 0.46 * 0.003 0.50 * 0.007 Ni 0.00 0.840 0.27 0.070 Cu 0.42 * 0.005 0.29 0.060 Zn 0.48 * 0.002 0.38 0.020 As 0.31 0.020 0.08 0.340 Se 0.05 0.410 0.06 0.430 Br 0.24 0.040 0.13 0.230 Cd 0.01 0.680 0.38 0.030 Ba 0.36 0.011 0.22 0.110 Pb 0.31 0.020 0.38 0.020 Total mass 0.03 0.450 0.51 0.003 The most significant associations are indicated by asterisks (*). Table 4. Effect of changes in the levels of ambient air particles on rat lung CL (cps/[cm.sup.2]). Treatment Lung CL Exposure to concentrated ambient particles Filtered air (5 hr) 24 [+ or -] 1 CAPs (a) (5 hr) 41 [+ or -] 4 ** CAPs (a) (5 hr)/room air (24 hr) 25 [+ or -] 4 Filtered air (5 hr)/room air (24 hr) 20 [+ or -] 2 Exposure to particle-free ambient air Room air (3 days) 27 [+ or -] 3 Filtered air (3 days) 16 [+ or -] 1 ** Filtered air (3 days)/room air 3 hr 20 [+ or -] 7 5 hr 32 [+ or -] 8 8 hr 32 [+ or -] 2 Values indicate mean [+ or -] SEM (n = 4-6). (a) Average CAPs concentration = 300 [+ or -] 60 [micro]g/[m.sup.3]. ** p < 0.001 compared with the control values. Table 5. Wet/dry ratios in the lung and heart of rats exposed to CAPs or filtered air. Lung Time of exposure Filtered air CAPs 1 hr 4.81 [+ or -] 0.06 4.77 [+ or -] 0.04 3 hr 4.72 [+ or -] 0.03 4.90 [+ or -] 0.04 *, (#) 5 hr 4.84 [+ or -] 0.04 4.92 [+ or -] 0.02 (#) 24 hr after 5 hr exposure 4.75 [+ or -] 0.08 4.8 [+ or -] 0.1 Heart Time of exposure Filtered air CAPs 1 hr 4.15 [+ or -] 0.02 4.11 [+ or -] 0.03 3 hr 4.20 [+ or -] 0.02 4.33 [+ or -] 0.04 *, (##) 5 hr 4.12 [+ or -] 0.04 4.29 [+ or -] 0.01 **, (##) 24 hr after 5 hr exposure 4.21 [+ or -] 0.02 4.27 [+ or -] 0.02 (##) Values indicate mean [+ or -] SE (n = 6-10). * p < 0.05 compared with control values for the same time of exposure. ** p < 0.002 compared with control values for the same day of exposure. (#) p < 0.02 compared with values after 1 hr exposure. (##) p < 0.0001 compared with values after 1 hr exposure. Table 6. Serum markers of tissue damage in rats exposed to CAPs or filtered air. Serum markers LDH (IU/mL) Time of exposure Filtered air CAPs 1 hr 440 [+ or -] 40 570 [+ or -] 100 3 hr 570 [+ or -] 60 700 [+ or -] 140 5 hr 530 [+ or -] 60 950 [+ or -] 180 *, (#) 24 hr after 5 hr exposure. 520 [+ or -] 20 580 [+ or -] 10 Serum markers CPK (IU/mL) Time of exposure Filtered air CAPs 1 hr 260 [+ or -] 40 360 [+ or -] 30 3 hr 420 [+ or -] 50 510 [+ or -] 60 (##) 5 hr 440 [+ or -] 30 550 [+ or -] 50 (##) 24 hr after 5 hr exposure. 420 [+ or -] 10 430 [+ or -] 10 Values indicate mean [+ or -] SE (n = 10). * p < 0.03 compared to the control values for the same time of exposure. (#) p < 0.03 compared to the values after 1 hr exposure. (##) p < 0.01 compared to the values after 1 hr exposure. Table 7. Activity of antioxidant enzymes in the lung and heart of rats exposed to CAPs or filtered air. Lung Activity Filtered air CAPs Cu/ZnSOD (U/mg protein) 38 [+ or -] 7 38 [+ or -] 10 MnSOD (U/mg protein) 6 [+ or -] 3 10 [+ or -] 2 * Catalase (mU/mg protein) 43 [+ or -] 4 55 [+ or -] 5 * Fumarase (U/mg protein) 0.17 [+ or -] 0.01 0.19 [+ or -] 0.01 Heart Activity Filtered air CAPs Cu/ZnSOD (U/mg protein) 170 [+ or -] 20 340 [+ or -] 60 * MnSOD (U/mg protein) 11 [+ or -] 1 15 [+ or -] 2 * Catalase (mU/mg protein) 0.28 [+ or -] 0.03 0.34 [+ or -] 0.02 Fumarase (U/mg protein) 1.7 [+ or -] 0.1 1.8 [+ or -] 0.1 Values indicate mean [+ or -] SE (n = 10). * p < 0.05 compared to control values.
Baeza-Squiban A, Bonvallot V, Boland S, Marano F. 1999. Airborne particles evoke an inflammatory response in human airway epithelium. Activation of transcription factors. Cell Biol Toxicol 15:375-380.
Barnard ML, Gurdian S, Turrens JF. 1993. Activated polymorphonuclear leukocytes increase low-level chemiluminescence of isolated perfused rat lungs. J Appl Physiol 75:933-999.
Beauchamp CO, Fridovich I. 1973. Isozymes of superoxide dismutase from wheat germ. Biochim Biophys Acta 317:50-64.
Boveris A, Cadenas E. 1997. Cellular sources and steady-state levels of reactive oxygen species. In: Oxygen, Gene Expression, and Cellular Function (L. Biadasz Clerch, Massaro, D.J., ed). New York:Marcel Dekker, Inc., 5-36.
Boveris A, Cadenas E, Reiter R, Filipkowski M, Nakase Y, Chance B. 1980. Organ chemiluminescence: noninvasive assay for oxidative radical reactions. Proc Natl Acad Sci USA 77:347-351.
Cadenas E, Sies H. 1984. Low-level chemiluminescence as an indicator of singlet molecular oxygen in biological systems. Methods Enzymol 105:221-231.
Calderon-Garciduenas L, Mora-Tiscareno A, Fordham LA, Chung CJ, Garcia R, Osnaya N, et al. 2001. Canines as sentinel species for assessing chronic exposures to air pollutants: part 1. Respiratory pathology. Toxicol Sci 61:342-355.
Churg A, Zay K, Li K. 1997. Mechanisms of mineral dustinduced emphysema. Environ Health Perspect 105(suppl 5):1215-1218.
Clarke RW, Catalano PJ, Coull B, Koutrakis P, Krishna Murty GG, Rice T, et al. 2000a. Age-related responses in rats to concentrated urban air particles (CAPs). Inhal Toxicol 12:73-84.
Clarke RW, Coull B, Reinisch U, Catalano P, Killingsworth C, Koutrakis P, et al. 2000b. Inhaled concentrated ambient particles are associated with hematological and bronchoalveolar lavage changes in canines. Environ Health Prespect 108:1179-1187.
Clerch LB, Massaro D. 1993. Tolerance of rats to hyperoxia. Lung antioxidant enzyme gene expression. J Clin Invest 91:499-508.
Crapo JD, McCord JM. 1976. Oxygen-induced changes in pulmonary superoxide dismutase assayed by antibody titrations. Am J Physiol 213:1196-1203.
Crawdford DR. 1999. Regulation of mammalian gene expression by reactive oxygen species. In: Reactive Oxygen Species in Biological Systems (Gilbert DL, Colton CA, eds). Washington, DC:Plenum Publisher Corp., 155-171.
Csonka C, Pataki T, Kovacs P, Muller SL, Schroeter ML, Tosaki A, et al. 2000. Effects of oxidative stress on the expression of antioxidative defense enzymes in spontaneously hypertensive rat hearts. Free Radic Biol Meal 29:612-619.
Dai J, Xie C, Chung A. 2001. Iron loading makes non-fibrinogenic titanium dioxide fibrinogenic [Abstract]. Am J Resp Crit Care Med. 163(5):A175.
Evelson P, Gonzalez-Flecha B. 2000. Time course and quantitative analysis of the adaptive responses to mild hyperoxia in the rat lung and heart. Biochim Biophys Acta 1523:209-216.
Ghio A J, Kim C, Devlin RB. 2001. Concentrated ambient air particles induce mild pulmonary inflammation in healthy human volunteers. Am J Resp Crit Care Med 162:981-982.
Godleski JJ, Verrier RL, Koutrakis P, Catalano P. 2000. Mechanisms of Morbidity and Mortality from Exposure to Ambient Air Particles. Cambridge MA:Health Effects Institute. Available: www.healtheffects.org/Pubs/ Godleski.pdf [accessed 29 April 2002].
Goldsmith CAW, Imrich A, Danaee H, Ning Y, Kobzik L. 1998. Analysis of air pollution particulate-mediated oxidative stress in alveolar macrophages. J Toxicol Environ Health 54:529-545.
Gonzalez-Flecha B, Cutrin JC, Boveris A. 1993. Time course and mechanism of oxidative stress and tissue damage in rat liver subjected to in vivo ischemia-reperfusion. J Clin Invest 91:456-464.
Halliwell B, Gutteridge JMC. 1990. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 186:1-88.
Imrich A, Ning YY, Koziel H, Coull B, Kobzik L. Lipopolysaccharide priming amplifies lung macrophage tumor necrosis factor production in response to air particles. Toxicol Appl Pharmacol 159:117-124 (1999).
Institute of Laboratory Animal Resources. 1996. Guide for the Care and Use of Laboratory Animals. 7th ed. Washington, DC:National Academy Press.
Jiang NF, Clarke RW, Rice TM, Streans R, Al-Mutairi E, Godleski JJ, et al. 2001. Compositionally distinct residual oil fly ashes elicit differential pulmonary leukocyte influx and cytokine mRNA expression [Abstract]. Am J Resp Crit Care Med 163(5):A175.
Jimenez LA, Thompson J, Brown DA, Rahman t, Antonicelli F, Duffin R, et al. 2000. Activation of NF-KB by P[M.sub.10] occurs via an iron-mediated mechanism in the absence of IKB degradation. Toxicol Appl Pharmacol 166:101-110.
Kadiiska MB, Mason RP, Dreher KL, Costa DL, Ghio AJ. 1997. In vivo evidence of free radical formation in the rat lung after exposure to an emission source of air pollution. Chem Res Toxicol 10:1104-1108.
Kaiser J. 2000. Evidence mounts that tiny particles can kill. Science 289:22-23.
Killingsworth CR, Alessandrini F, Krishna Murty GC, Catalano P J, Paulauskis JD, Godleski JJ. 1997. Inflammation, chemokine expression, and death in monocrotaline-treated rats following fuel coal ash inhalation. Inhal Toxicol 9:541-565.
Kodavanti UP, Schladweiler MC, Richards JR, Costa DL. 2001. Acute lung injury from intratracheal exposure to fugitive residual oil fly ash and its constituent metals in normo- and spontaneously hypertensive rats. Inhal Toxicol 13:37-54.
Lissi E, Videla L, Gonzalez-Flecha B, Giulivi C, Boveris A. 1991. Metabolic regulation in oxidative stress: an overview. In: Oxidative Damage and Repair (Davies KJA, ed). Oxford:Pergamon Press, 444-448.
Lores Arnaiz S, Llesuy S. 1993. Oxidative stress in mouse heart by antitumoral drugs: a comparative study of doxorubicin and mitoxantrone. Toxicol 77:31-38.
Lowry OH, Rosembrough AL, Farr AL, Randall R. 1951. Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275.
Madden MC, Thomas MJ, Ghio AJ. 1999. Acetaldehyde (CH3CHO) production in rodent lung after exposure to metal-rich particles. Free Radic Biol Med 26:1569-1577.
McCord JM, Fridovich I. 1969. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244:6049-6055.
Monn C, Becker S. 1999. Cytotoxicity and induction of proinflammatory cytokines from human monocytes exposed to fine (P[M.sub.2.5]) and coarse particles (P[M.sub.10-2.5])in outdoor and indoor air. Toxicol Appl Pharmacol 155:245-252.
Morton RL, Das KC, Guo XL, Ikle DN, White CW. 1999. Effect of oxygen on lung superoxide dismutase activities in premature baboons with bronchopulmonary dysplasia. Am J Physiol 276:L64-L74.
Mossman BT. 2000. Mechanisms of action of poorly soluble particulates in overload-related lung pathology. Inhal Toxicol 12:141-148.
Murphy SA, BeruBe KA, Pooley FD, Richards RJ. 1998. The response of lung epithelium to well characterised fine particles. Life Sci 62:1789-1799.
Nadadur SS, Schladweiler MC, Kodavanti UP. 2000. A pulmonary rat gene array for screening altered expression profiles in air pollutant-induced lung injury. Inhal Toxicol 12:1239-1254.
NIH. 2000. Principles for the Use of Animals. Bethesda, MD:National Institutes of Health.
Nel AE, Diaz-Sanchez D, Li N. 2001. The role of particulate pollutants in pulmonary inflammation and asthma: evidence for the involvement of organic chemicals and oxidative stress. Curr Opin Pulm Med 7:20-26.
Nelson D, Kiesow LA. 1972. Enthalpy of decomposition of hydrogen peroxide by catalase at 25 degrees C (with molar extinction coefficients of [H.sub.2][O.sub.2] solutions in the UV). Anal Biochem 49:474-478.
Plopper C6, Duan X, Buckpitt AR, Pinkerton KE. 1994. Dose-dependent tolerance to ozone. IV. Site-specific elevation in antioxidant enzymes in the lung of rats exposed for 90 days or 20 months. Toxicol Appl Pharmacol 127:124-131.
Prahalad AK, Soukup JM, Inmon J, Willis R, Ghio A J, Becker S, et al. 1999. Ambient air particles: effects on cellular oxidant radical generation in relation to particulate elemental chemistry. Toxicol Appl Pharmacol 158:81-91.
Racker E. 1950. Spectrophotometric measurements of the enzymatic formation of fumaric and cis-aconitic acids. Biochim Biophys Acta 4:211-214.
Rohrdanz E, Schmuck G, Ohler S, Tran-Thi QH, Kahl R. 2001. Changes in antioxidant enzyme expression in response to hydrogen peroxide in rat astroglial cells. Arch Toxicol 75:150-158.
Saldiva PHN, Clarke RW, Streans RC, Lawrence J, Koutrakis P, Godleski JJ. 2001. Pulmonary inflammation in rats exposed to concentrated ambient particles (CAPs) is associated with combustion components [Abstract]. Am J Resp Crit Care Med 163(5):A171.
Shukla A, Timblin C, BeruBe K, Gordon T, McKinney W, Driscoll K, et al. 2000. Inhaled particulate matter causes expression of nuclear factor (NF)-[kappa]B-related genes and oxidant-dependent NF-KB activation in vitro. Am J Respir Cell Mol Biol 23:182-187.
Sioutas C, Koutrakis P, Burton RM. 1995. A technique to expose animals to concentrated fine ambient aerosols. Envrion Health Perpect 103:172-177.
Stringer B, Kobzik L. 1998. Environmental particulate-mediated cytokine production in lung epithelial cells (A549): role of preexisting inflammation and oxidant stress. J Toxicol Environ Health 55:31-44.
Tannahill CL, Stevenot SA, Eaker EY, Sallustio JE, Nick HS, Valentine J. 1997. Regulation of superoxide dismutase in primary cultures of rat colonic smooth muscle cells. Am J Physiol 272:G1230-1235.
Timblin C, BeruBe K, Churg A, Driscoll K, Gordon T, Hemenway D, et al. 1998. Ambient particulate matter causes activation of the c-jun kinase/stress-activated protein kinase cascade and DNA synthesis in lung epithelial cells. Cancer Res 58:4543-4547.
Turrens JF, Giulivi C, Pinus CR, Lavagno C, Boveris A. 1988. Spontaneous lung chemiluminescence upon paraquat administration. Free Radic Biol Med 5:319-323.
Valentine JF, Nick HS. 1999. Inflammatory regulation of manganese superoxide dismutase, in: Reactive Oxygen Species in Biological Systems (Gilbert DL, Cotton CA, eds). Washington, DC:Plenum Publisher Corp., 173-189.
Venables WN, Ripley BD. 1994. Modern Applied Statistics with S-Plus. New York:Springer.
Watkinson WP, Nolan JP, Kodavanti UP, Schladweiler MCJ, Evansky PA, Lappi ER, et al. 2001. Effects of inhalation of soluble metallic constituents of particulate matter with preexposure and/or concurrent exposure to ozone on cardiovascular and thermoregulatory parameters in awake rats [Abstract]. Am J Resp Crit Care Med 163(5):A312.
Address correspondence to B. Gonzalez-Flecha, Physiology Program, Department of Environmental Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115 USA. Telephone: (617) 432-1277. Fax: (617) 432-0014. E-mail: email@example.com
Data presented in this article were presented in part at the 2001 FASEB meeting held 31 March-4 April 2001 in Orlando, Florida.
We thank E. Diaz for help in setting up and carrying out the ROFA exposures, R. Streans for carrying out the X-ray fluorescence analysis of carbon black particles, and L. Kobzik, J. Godleski, and P. Koutrakis for many insightful discussions.
S.A.G. is supported in part by a fellowship from the FAPESP (Brazil). This work was supported by grants from the National Institutes of Health (P01 ES08129-03 and P50 ES00002-39) and by the Francis Families Foundation.
Received 12 December 2001; accepted 17 January 2002.
Sonia A. Gurgueira, (1) Joy Lawrence, (1) Brent Coull, (2) G. G. Krishna Murthy, (1) and Beatriz Gonzalez-Flecha (1)
(1) Department of Environmental Health, and (2) Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts, USA
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|Date:||Aug 1, 2002|
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