Spectrochimica Acta Part A 60 (2004) 1371–1377
ESR investigation of the oxidative damage in lungs caused by asbestos and air pollution particles M.B. Kadiiska a,∗ , A.J. Ghio b , R.P. Mason a a
National Institutes of Environmental Health Sciences, National Institutes of Health, MD F0-02, P.O. Box 12233, Research Triangle Park, NC 27709, USA b Environmental Protection Agency, Research Triangle Park, NC 27709-2233, USA Received 15 July 2003; accepted 19 October 2003
Abstract Exposure to asbestos and air pollution particles can be associated with increased human morbidity and mortality. However, the molecular mechanism of lung injuries remains unknown. It has been postulated that the in vivo toxicity results from the catalysis of free radical generation. Using electron spin resonance (ESR) in conjunction with the spin trap ␣-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) we previously investigated in vivo free radical production by rats treated with intratracheal instillation of asbestos (crocidolite fibers) and an emission source air pollution particle (oil fly ash). In this report we compare the effect of two different exposures on the type of free radicals they induce in in vivo animal model. Twenty-four hours after the exposure, ESR spectroscopy of the chloroform extract from lungs of animals exposed to either asbestos or oil fly ash gave a spectrum consistent with a carbon-centered radical adduct (aN = 15.01 G and aH = 2.46 G). To test whether free radical formation occurred in vivo and not in vitro, a number of control experiments were performed. Combinations (both individually and together) of asbestos or oil fly ash and 4-POBN were added to lung homogenate of unexposed rats prior to chloroform extraction. No detectable ESR signal resulted. To exclude the possibility of ex vivo free radical generation, asbestos or oil fly ash was added to lung homogenate of an animal treated with 4-POBN. Also, 4-POBN was added to lung homogenate from rats instilled with asbestos or oil fly ash. Neither system produced radical adducts, indicating that the ESR signal detected in the lung extracts of the treated animals must be produced in vivo and not ex vivo or in vitro. In conclusion, ESR analysis of lung tissue demonstrated that both exposures produce lipid-derived radical metabolites despite their different composition and structure. Analogously, both exposures provide evidence of in vivo enhanced lipid peroxidation. Furthermore, it is concluded that without the presence of a spin-trapping agent, no free radical metabolites could be detected directly by ESR in either exposure. © 2003 Published by Elsevier B.V. Keywords: ESR; Spin trap; Asbestos; Oil fly ash; Free radicals in vivo
1. Introduction The toxicity of a wide variety of xenobiotics appears to be related to their ability to induce oxidative stress, characterized by increased free radical production. The free radical species formed in cells by exogenous agents of endogenously generated responses can readily attack proteins, DNA, and lipids, resulting in their loss of function and damage. For example, it has been postulated that the in vivo toxicity of asbestos results from its catalysis of free radical generation. ∗ Corresponding author. Tel.: +1-919-541-0201; fax: +1-919-541-1043. E-mail address:
[email protected] (M.B. Kadiiska).
1386-1425/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/j.saa.2003.10.035
Inhalation of asbestos can be associated with lung inflammation, fibrosis, and genotoxicity in humans [1]. The mechanisms responsible for this injury are not known. In both acellular and cellular systems, fibrous silicates generate oxygen- and nitrogen-centered free radicals including superoxide, hydroxyl radical, and nitric oxide [2,3]. This catalysis of free radicals by asbestos is postulated to participate in the pathogenesis of disease which occurs after their inhalation [4]. Epidemiologic studies have established an association between exposure to air pollution particles and human mortality and morbidity. Elevated levels of air pollution can affect airway reactivity with decrements in several indices of pulmonary function [5,6] especially in those patients with preexisting asthma. Air pollution particles are also associated
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with increased incidences of both pulmonary infections and hospitalization for respiratory disease [7–9]. These health effects are observed at particle concentrations below the current air quality standard of 150 g m−3 set to protect public health. The mechanism(s) of lung injury after exposure to air pollution particles and asbestos are not known. Injury has been postulated to be mediated by ultrafine particles [10] biological agents (e.g., endotoxin) [11], acid aerosols [12], and polyaromatic hydrocarbons [13]. Oxidant generation catalyzed by metals associated with air pollution particles could also account for lung injury following exposure to air pollution particles. The in vitro generation of oxygenderived free radicals by both emission source and ambient air pollution particles has been documented [14,15]. The in vivo formation of free radicals in tissue after exposure to environmental toxins has always been a challenge and difficult to demonstrate. Spin trapping continues to be the only approach available to detect by ESR short-lived reactive free radicals at low concentrations in biological systems. The use of spin-trapping compounds for the study of radicals having extremely high rates of reactivity in biological reactions appears to have the potential for the detection and quantitation of radicals that would not be possible by other methods. The advantage of spin trapping is to convert unstable or short-lived free radicals into more stable aminoxyl radicals for which ESR spectra can be easily reported. It is well known that the technique of spin trapping involved the addition of a primary free radical across the double bond of a diamagnetic compound (the spin trap) to form a radical adduct more stable than the primary free radical. This technique involves the indirect detection of primary free radicals that cannot be directly observed by conventional ESR due to low steady state concentrations or to very short relaxation times, which lead to very broad lines. By using ESR spin-trapping technique, we tested the hypothesis that exposure to an emission source air pollution particle and asbestos is indeed associated with in vivo free radical production. In addition, we attempted to characterize the radicals generated during the two different exposures and to compare the effect of two different exposures on the type of free radicals they induce in in vivo animal model.
power plant which was burning a low sulfur number six residual oil (collection temperature of 204 ◦ C). Vanadium(IV) sulfate oxide (VOSO4 ·4H2 O) was acquired from Alfa (Ward Hill, MA, USA). All other materials were obtained from Sigma unless otherwise specified. The animals employed in all studies were 60-day-old, male Sprague-Dawley rats (Charles River Breeding Laboratories, Raleigh, NC, USA). One day, after instillation of either crocidolite (500 g per rat) or oil fly ash (500 g per rat) rats were intraperitonally injected with 8 mmol kg−1 body weight ␣-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN). After 1 h, the animals were anesthetized with 25 mg kg−1 body weight pentobarbital (Abbott Laboratories, North Chicago, IL) intraperitoneally and euthanized by exanguination. The lungs were excised and homogenized in 1.0 ml 50 mM 2,2 -dipyridyl, 3.0 ml of 2:1 chloroform:methanol, 1.0 ml chloroform, and 1.0 ml deionized water. After centrifugation at 1000 × g for 10 min (Beckman TJ-6), the chloroform layer was isolated. ESR spectra were recorded immediately at room temperature using a quartz flat cell and a Varian E-109 ESR spectrometer equipped with a TM110 cavity and operating a 9.33 GHz, 20 mW microwave power, and 100 kHz modulation frequency. Spectra were recorded on an IBM-compatible computer interfaced with the spectrometer. The ESR spectral simulations were performed using an automatic optimization procedure (http://epr.niehs.nih.gov). In addition to animals exposed to saline, controls included rats intratracheally instilled with 500 g crocidolite or oil fly ash without the injection of 4-POBN. Animals (n = 3) were also instilled with a “synthetic oil fly ash”, which is a mixture of vanadyl, nickel, and iron(III) sulfates in concentrations approximately equivalent to those included in 500 g of oil fly ash (0.5 ml of the synthetic oil fly ash, which included a mixture of 3700 M VOSO4 , 640 M NiSO4 , and 630 M Fe2 (SO4 )3 ). This synthetic oil fly ash was made up in normal saline and had a pH value of 4.32. Twenty-four hours later, 4-POBN was provided, the lungs were excised and homogenized, and the ESR spectra were recorded. Finally, this was repeated after the animals were instilled with 0.5 ml of either 1.0 mM VOSO4 (n = 3), 1.0 mM NiSO4 (n = 3), or 1.0 mM Fe2 (SO4 )3 (n = 3) in normal saline (pH values of 3.82, 4.47, and 2.75, respectively).
2. Materials and methods
2.1. In vitro ESR investigation
The asbestos used was crocidolite from the National Institute of Environmental Health Sciences (Research Triangle Park, NC) with a surface area of 8.7 ± 1.0 m2 g−1 (Bruneur–Emmett–Teller nitrogen adsorption isotherm). The air pollution particle used was oil fly ash which had a mass median aerodynamic diameter of 1.95 ± 0.18 m and was acquired from Southern Research Institute (Birmingham, AL, USA). It was collected in Florida using a Tefloncoated fiberglass filter downstream from the cyclone of a
To provide further controls, in ESR studies 500 g crocidolite and 8 mmol kg−1 body weight 4-POBN were added to lung tissue obtained from unexposed animals and homogenized in 1.0 ml 50.0 mM 2,2 -dipyridyl, 3.0 ml 2:1 chloroform:methanol, 1.0 ml chloroform, and 1.0 ml deionized water. For experiments involving in vitro generation of 4-PBN/ethyl or 4-POBN/pentyl radical adduct, incubation mixtures were prepared by oxidizing the corresponding alkylhydrazine with CuCl2 as described elsewhere [16,17].
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A mixture of carbon-centered fatty acid-derived radical adducts was prepared by the peroxidation of arachidonic or linoleic acid by soybean lipoxygenase [16,18]. In a typical experiment, 1.5 ml incubation mixture was added to lung homogenates from non-treated rats, and ESR analysis of the lipid extract of lung tissue was performed as described earlier. 2.2. Statistical analyses Data are expressed as mean values ± standard deviations except for ESR spectra. Differences between multiple groups were compared using analysis of variance. Two tailed tests of significance were used. The post hoc test employed was Scheffe’s. Significance was assumed at P < 0.05 [19].
3. Results Gravimetric determinations of oil fly ash content demonstrated that 88.7 ± 1.2% of the dust was soluble in water (n = 6, w/w). There was little carbon in the oil fly ash, while mineral ash analysis revealed high concentration of vanadium, nickel, and iron compounds and sulfate. Watersoluble concentrations of vanadium, nickel, and iron in the dust accounted for 78.9, 97.0, and 80.4%, respectively, of the total concentrations of these metals in the oil fly ash. These metals are likely to exist as soluble sulfates and ammonium sulfates. That metal retained in the insoluble component is probably mineral oxides such as V2 O5 and Fe2 O3 . Twenty-four hours after instillation of oil fly ash, prominent six-line radical adduct ESR spectra could be detected in samples of lung lipid extracts (Fig. 1A). The injection of saline resulted in a much weaker spectrum (Fig. 1B). The instillation of the air pollution particle into rats without injected 4-POBN did not yield detectable radical species in lipid extracts from the lungs, confirming the dependence of radical generation on the availability of the spin trap (Fig. 1C). This spectrum was simulated using a computer program (http://epr.niehs.nih.gov). The hyperfine coupling constants for the 4-POBN radical adducts were aN = 15.0 G and aH = 2.5 G (Fig. 2). Lipid extracts of lungs acquired from rats exposed to the soluble component of the oil fly ash demonstrated an ESR spectrum identical to those animals instilled with the air pollution particle (Fig. 3A). The coupling constants were aN = 15.01 G and aH = 2.46 G. However, exposure of the animals to the insoluble component was associated with a much smaller ESR signal (Fig. 3B). The ESR spectrum of lung lipid extracts from animals instilled with synthetic oil fly ash which is a mixture of vanadyl, nickel, and iron(III) sulfates resulted in the detection of a signal comparable to that exhibited by the soluble fraction of the oil fly ash (Fig. 3C). Finally, individual metal sulfates (0.5 ml of 1.0 mM solutions) were instilled into the animals. Saline (Fig. 4A) and
Fig. 1. (A) ESR spectrum of 4-POBN radical adducts detected in lipid extracts of lungs 24 h after intratracheal instillation of oil fly ash (B). Same as in (A), but rats were instilled with saline. (C) Same as in (A), but rats were not administered 4-POBN. Instrumental conditions: microwave power, 20 mW; modulation amplitude, 1.33 G; time constant, 1 s; and scan rate, 5 G min−1 .
nickel sulfate (Fig. 4D) resulted in small ESR signals, while the spectra of lipid extract after vanadium (Fig. 4B) and iron sulfates (Fig. 4C) indicated a significant production of radical adducts. ESR spectra after instillation of vanadyl sulfate were more intense than those acquired from animals exposed to ferric sulfate, but otherwise similar. Twenty-four hours after instillation of crocidolite, prominent six-line ESR spectra could be detected in samples
Fig. 2. (A) ESR spectrum of 4-POBN radical adducts detected in lipid extracts of lungs 24 h after intratracheal instillation of oil fly ash (500 g per rat) and 1 h after intraperitoneal administration of 4-POBN (8 mmol kg−1 ). (B) Simulated spectra of (A). Instrumental conditions: microwave power, 20 mW; modulation amplitude, 1.33 G; time constant, 1 s; and scan rate, 5 G min−1 .
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Fig. 3. (A) ESR spectrum of radical adducts detected in lipid extract of lungs 24 h after intratracheal instillation of the soluble fraction of the air pollution particle. (B) ESR spectrum of radical adducts detected in lipid extract of lungs 24 h after intratracheal instillation of the insoluble fraction of the air pollution particle. (C) ESR spectrum of radical adducts detected in lipid extract of lungs 24 h after intratracheal instillation of a mixture of vanadyl, nickel, and iron(III) sulfates reflecting the concentrations of these metals in the oil fly ash. Instrumental conditions: microwave power, 20 mW; modulation amplitude, 1.33 G; time constant, 1 s; and scan rate, 5 G min−1 .
of lipid extracts as well (Fig. 5A). This same spectrum was detected at both 7 and 30 days after asbestos exposure. The injection of saline resulted in a much weaker spectrum (Fig. 5B). The instillation of crocidolite into rats
Fig. 4. ESR spectrum of radical adducts detected in lipid extract of lungs 24 h after intratracheal instillation of (A) 0.5 ml of saline, (B) 1.0 mM vanadyl sulfate, (C) 1.0 mM iron(III) sulfate, and (D) 1.0 mM nickel sulfate. Instrumental conditions: microwave power, 20 mW; modulation amplitude, 1.33 G; time constant, 1 s; and scan rate, 5 G min−1 .
Fig. 5. (A) ESR spectrum of 4-POBN radical adducts detected in lipid extracts of lungs 24 h after intratracheal instillation of crocidolite and 1 h after intraperitoneal administration of 4-POBN. (B) Same as in (A), but rats were not instilled with crocidolite. (C) Same as in (A), but rats were not administered 4-POBN. Instrumental conditions: microwave power, 20 mW; modulation amplitude, 1.33 G; time constant, 1 s; and scan rate, 5 G min−1 .
without injected 4-POBN did not yield detectable radical adducts in lipid extracts from the lungs, confirming the dependence of radical detection on the availability of the spin trap (Fig. 5C). The ESR spectrum shown in Fig. 5A was simulated using again the computer program (http://epr.niehs.nih.gov). The hyperfine coupling constants for the 4-POBN radical adducts were aN = 15.01 G and aH = 2.46 G. Metal cations available in either lung homogenate or lipid extracts could catalyze the generation of the radicals detected. To determine whether the 4-POBN adduct was formed in vivo in the lungs of the animals or ex vivo during sample preparation, a series of control experiments were performed. Since 2,2’-dipyridyl inhibits ex vivo reactions initiated by Fe2+ , lung homogenate in chloroform/methanol was diluted with 50 mM solution of this chelator in place of deionized water. The intensity of the ESR signals from lipid extracts of lungs prepared with 2,2 -dipyridyl (Fig. 6B) was decreased only modestly relative to spectra obtained while the metal chelator was omitted (Fig. 6A). Experiments with control rats demonstrated no differences in ESR spectra regardless of the presence or absence of 2,2 -dipyridyl Figs 6C and D). Since 2,2 -dipyridyl decreased the intensity of the ESR signals in those lipid extracts of lungs from rats instilled with crocidolite, 50 mM of the chelator was employed rather than deionized water to dilute lung chloroform/methanol homogenate. All in vivo data presented were subsequently obtained from samples prepared with 2,2 -dipyridyl included in homogenates and solutions. Rats instilled with crocidolite and injected intraperitioneally with
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Fig. 6. (A) ESR spectrum of radical adducts detected in lipid extract of lungs 24 h after intratracheal instillation of crocidolite and 1 h after intraperitoneal administration of 4-POBN. (B) Same as in (A), but lipid extract of lungs was done in the presence of 2,2-dipyridyl. (C) Same as in (A) but without crocidolite instillation. (D) Same as in (C), but with 2,2-dipyridyl. Instrumental conditions: microwave power, 20 mW; modulation amplitude, 1.33 G; time constant, 1 s; and scan rate, 5 G min−1 .
deferoxamine had no change in lavage neutrophils and protein concentrations relative to saline treatment. Similarly, the deferoxamine treatment did not diminish the ESR signal relative to those animals injected with saline (data not shown). Radical adducts of 4-POBN could also result from ex vivo reactions of metal ions associated with either crocidolite or other trace metals. To test whether free radical formation occurred in vitro, combinations (both individually and together) of 500 g crocidolite and 8 mmol kg−1 body weight 4-POBN were added to lung homogenate of unexposed rats prior to lipid extraction. No detectable ESR signal resulted (data not shown).
4. Discussion The presentation of this ESR spectra provides evidence of an in vivo oxidant generation by an air pollution particle and asbestos. Instillation of the soluble fraction of the oil fly ash in an animal provided an ESR spectrum similar to that of the emission source air pollution particle, while in vitro oxidant generation (i.e., thiobarbituric acid-reactive products of deoxyribose) supported oxygen-based free radicals as the dominant species [14]. This suggests that metals • included in the oil fly ash catalyzed oxidants (e.g., OH) which initiated a lipid peroxidation in the lower respiratory tract of the animal. The detection and identification of a chloroform-soluble 4-POBN radical adduct following croci-
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dolite instillation in the rat lung was based on the hyperfine coupling constants (aN = 15.01 G and aH = 2.46 G) of authentic ethyl and pentyl radical adducts of 4-POBN obtained in vitro [20]. The 4-POBN radical adducts detected by ESR after instillation of the air pollution particle are very similar, if not identical, to ethyl and pentyl radical adducts. Therefore, it is proposed that in this in vivo system, the spin trap reacted with ethyl and pentyl radicals (or structurally similar radicals) to produce a radical adduct with hyperfine coupling constants aN = 15.0 G and aH = 2.5 G. Alkyl radicals are produced during the process of lipid peroxidation [21–25]. These radicals result from the reductive decomposition of lipid peroxides. The carbon-centered alkyl radicals, which result from the -scission of the lipid alkoxyl radicals, can either react with molecular oxygen to produce peroxyl radicals or abstract a hydrogen atom to form ethane and pentane, which are employed as indicators of lipid peroxidation [26,27]. Emission source particles of both natural and anthropogenic origin can include soluble metal salts and insoluble components that may have the capacity to complex metals at the surface. Metals which exist in more than one stable valence state can participate in electron transfer reactions and subsequently possess a potential to generate oxidants. Among metals which can assume two stable valence states, the first-row transition metals titanium, vanadium, chromium, manganese, iron, cobalt, nickel, and copper are found in greatest concentration in air pollution particles [28]. Exposures to these metals can be associated with oxidative stress [29–32]. Soluble metal salts in greatest quantities in atmospheric particulates are likely to include acid sulfites/sulfates [33]. Insoluble inorganic components with a capacity to complex metals at the solid interface are predominantly oxides including SiO2 , Fe2 O3 , and A12 O3 . The metal compounds responsible for free radical formation associated with exposure to the oil fly ash were contained in the water-soluble fraction of this emission source air pollution particle. Vanadium, nickel, and iron sulfates are in high concentration in the oil fly ash and are included in the water-soluble fraction of the particle [34–36]. Human exposure to oil fly ash results in a lung injury in which vanadium compounds are proposed to be responsible [37]. The intratracheal instillation of a vanadium compound produced an ESR signal comparable to those seen after exposure of the animal to oil fly ash, its soluble fraction, and the mixture of metal sulfate. These results suggest a significant role for vanadium in both oxidative stress and lung injury after exposure to oil fly ash. Asbestos contains chemically reactive iron [37] and, in the presence of hydrogen peroxide, generates hydroxyl radical both in vitro [38] and in vivo [39]. In vitro lipid peroxidation after exposure to these fibrous silicates is irondependent and can be inhibited by metal chelation [40–42]. The failure of deferoxamine to either eliminate or diminish the ESR signal does not prove that iron does not participate in in vivo damage. Several injuries associated with metal-generated oxidants are not affected by this chelator
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[43,44] and the cellular uptake and intracellular distribution of this compound may preclude its availability to sites of iron-catalyzing free radical generation. In vitro lipid peroxidation after exposures to fibrous silicates occurs in both acellular and cellular systems [40–42]. Metal associated with asbestos can catalyze lipid peroxidation in systems which include only fatty acids and liposomes. In addition, these mineral oxides stimulate accumulated alveolar macrophages and neutrophils to produce an abundance of oxygen-based free radicals. Both mechanisms of oxygen activation together could initiate lipid peroxidation in vivo. We do not totally exclude a role for activated phagocytes in radical adduct formation but the fact that cyclophosphamide decreased lavage neutrophils but not the magnitude of the ESR spectrum suggests that their role cannot be that important in radical adduct formation. After inhalation of crocidolite by rats, in vivo lipid peroxidation was demonstrated in both lavage cells and fluid [45]. Similarly, products of lipid peroxidation can be detected in lung tissue after intratracheal instillation of asbestos in a rat [46]. Finally, humans exposed to asbestos can similarly demonstrate elevated levels of thiobarbituric acid-reactive substances, supporting increased lipid peroxidation [47]. Reactive oxygen and nitrogen species have been implicated in the pathogenesis of asbestos fibers-associated pulmonary diseases [48–50]. Although it has been reported that mitochondric-regulated death pathway mediates asbestosinduced alveolar epitheial cell apoptosis [51], the molecular basis of asbestos-induced lung injury is not fully understood [52]. The mechanism of tissue injury after exposure to oil fly ash is not known either [53,54]. The peroxidation of endogenous phospholipids in biological membranes is considered to play an important role in mediating pathological processes and damage [55]. Asbestos has appreciable amounts of iron within its lattice structure which can act as a catalyst for the generation of oxygen radicals [56]. Similarly, oil fly ash has iron in addition to a number of other metals. An important end result of oxygen free radical damage is the peroxidation of polyunsaturated fatty acids which can lead to the enhanced lipid peroxidation and formation of lipid peroxidation products [57]. We conclude that instillation of crocidolite and oil fly ash into the rat induce free radical generation in the lungs that can be detected by ESR. The 4-POBN adducts detected by ESR are consistent with carbon-centered radicals and provide evidence of in vivo enhanced lipid peroxidation associated with asbestos and oil fly ash exposures. In addition, comparison of the free radical generation in lungs after the two different exposures described here and reported previously [20,58] showed interesting similarities. Both exposures produce lipid-derived radical metabolites despite their different composition and structure. Analogously, both exposures provide evidence of in vivo enhanced lipid peroxidation. Furthermore, it is concluded that
without the presence of a spin-trapping agent, no free radical metabolites could be detected directly by ESR in either exposure.
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