* Department of Pharmacology, UCLA School of Medicine, Center for the Health Sciences, Los Angeles, California 90095-1735; Department of Environmental Health Sciences, UCLA School of Public Health, Los Angeles, California 90095-1772; and
UCLA Interdepartmental Program in Molecular Toxicology, Center for the Health Sciences, Los Angeles, California 90095-1772
Received January 19, 2004; accepted June 7, 2004
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ABSTRACT |
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Key Words: diesel exhaust particles; DNA cleavage; free radical; oxidative stress.
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INTRODUCTION |
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DEP are heterogeneous species containing numerous organic and inorganic compounds (BéruBé et al., 1999; Williams and Chock, 1980
). Although many of the individual components of DEP are capable of performing biological chemistry that is potentially deleterious to cells, it seems likely that the toxicity of DEP may also be due to the combined chemistry of the proximal chemical entities contained within the particle. That is, the localization of redox active organics and inorganics contained in a carbon core matrix can lead to a chemically active catalytic particle whose overall chemical reactivity may be greater than the sum of its individual components. Previous researchers have, for example, found that intratracheal instilled DEP in mice leads to the formation of the potent oxidant hydroxyl radical (HO·) (Han et al., 2001
). Moreover, DEP have been shown to be capable of generating species with HO·-like reactivity in the presence of a reductant (Vogl and Elstner, 1989
). Significantly, DEP have been found to be capable of crossing airway epithelial cell membranes, taking residence in cells and eliciting an inflammatory response (Boland et al., 1999
). More recently, it has been demonstrated that ultrafine (<100 nm) particulate matter (PM) are capable of entering both lung epithelial cells and macrophages, gaining access to intracellular targets (Li et al., 2003
). These reports indicate that the size distribution of the airborne particulate matterincluding DEPmay be an important aspect of their toxicology since ultrafine PM are capable of traversing cell membranes leading to mitochondrial damage. Thus, we have examined some of the chemical properties of DEP so we can begin to define them chemically and increase our understanding of their inherent toxicity.
In this study, we specifically address the possibility that DEP are capable of catalyzing the generation of ROS that can lead to a toxic insult to exposed cells. Since abnormal generation of ROS is known to be deleterious to cells, DEP-catalyzed generation of ROS may be an important aspect of the toxicity associated with DEP exposure. Herein, we find that DEP are capable of catalyzing the generation of ROS, a process that can lead to, among other things, DNA damage. Of particular note, DEP whose easily extractable components have been removed by organic solvent or acid washes, maintain the ability to catalyze the generation of ROS, indicating an inherent activity of the particle itself.
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MATERIALS AND METHODS |
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Preparation of native DEP, organic washed DEP, and acid washed DEP. Preparation of DEP suspensions used in the O2-consumption assays was accomplished by adding the appropriate amount of DEP to a volume of 1 M potassium phosphate buffer (pH 7.4). The heterogenous suspension was then subjected to sonication for 2 min using a Branson Sonifier 250 (Danbury, CT) set at a 10% duty cycle and output control of 2. The sonicated suspensions could then be injected via syringe into the reactions vessels (vide infra). Methylene chloride washed DEP were prepared as follows. DEP were extracted with dichloromethane using a 1:5 (w/v) ratio of DEP to solvent. The suspension was then sonicated for 30 min using a 125W water bath sonicator. The sample was then centrifuged for 10 min at 850 x g at 4°C. The DEP pellet was then separated from the organic extract and the extract dried using a nitrogen gas stream. The extract residue was resuspended in 0.1 ml of DMSO for further analysis. The DEP after extraction were air-dried and resuspended in 1 M of phosphate buffer. Extraction of DEP with aqueous acid was performed as follows. Five mg of DEP were suspended in 1 ml of 1 M HCl and vortexed for 30 s. The sample was then centrifuged for 3 min at 13,600 x g. A disposable Pasteur pipette was used to remove the supernatant. Particles were then washed three times with 1 ml of deionized water (treated with Chelex 100 resin to remove trace metals) using the same procedure as described above. The acid washed DEP were resuspended in 1 M phosphate buffer, pH 7.4 (treated with Chelex 100) and vortexed for 30 s. Since extraction by methylene chloride or acid may decrease the mass of the DEP, in all cases, experiments performed with extracted particles represents an exposure to a 5 mg-equivalent dose of DEP.
Measurement of NADH, glutathione, Trolox, ascorbic acid, and DTT oxygen consumption in the presence of DEP. A Clarke electrode (Yellow Springs Model 5300 Biological Oxygen Monitor, Yellow Springs, OH) was used to measure DEP-dependent oxygen consumption by various reductants. A 10 ml three-necked, round bottom flask was filled entirely with air-saturated 1 M phosphate buffer (pH 7.4). In one neck of the flask, the Clarke electrode was inserted using a gas-tight adapter such that the electrode surface was in contact with the solution. The two other necks of the flask were capped with rubber septa through which solutions could be injected. No headspace gas was present. When solutions were injected into the flask through one of the septa, an open needle was inserted through the other septa to allow the displaced solution to leave the flask. The solution was stirred throughout the experiment using a magnetic stirrer. Reducing agents were injected into the flask using a syringe to final concentrations of 250 µM or 500 µM. Oxygen consumption was then monitored for 10 min. After 10 min, 5 mg of DEP in 1 ml of 1 M potassium phosphate buffer was injected into the flask and the rate of O2 consumption monitored. The rate of O2 consumption was determined by monitoring the decrease in the detector response over time and by assuming the initial concentration of O2 in air-saturated buffer to be 245 µM. O2 consumption rate is reported as µM/min/mg DEP.
Plasmid DNA nicking assay. DEP-dependent oxidative damage to DNA was assessed by using a plasmid DNA nicking assay. Briefly, each reaction mixture (final volume of 20 µl) contained 200 ng pUC 19 DNA in 100 mM potassium phosphate buffer, 500 µg/ml of DEP (native DEP, organic washed DEP and acid washed DEP), and 500 µM of ascorbic acid. The sample was incubated with agitation (150 RPM) for 2 h at room temperature. After incubation, 4 µl of a Blue/Orange 6X loading dye was added to the sample to stop the reaction. The samples were then loaded onto a 1.3 % agarose gel containing ethidium bromide and run at 100 V for 1 h at room temperature in 1 M Tris-acetate-EDTA buffer (TAE). Typhoon 9410 (blue laser module) with ImageQuant software (Amersham Biosciences, Piscataway, NJ) was used to perform densitometric analysis of the separated bands and to quantify the amount of supercoiled and open circular DNA. The results were expressed as the percent of open circular form over the sum of supercoiled and open circular forms (linear DNA was not observed). Since ethidium bromide binds "nicked" DNA better than supercoiled DNA, a correction factor for the supercoiled form of 1.4 fold was used to account for these differences (for example, Ohshima et al., 1999). Experiments were performed in triplicate.
Electron paramagnetic resonance (EPR) spectrometry. EPR spectra were recorded using a Bruker ER 200 D-SRC 9/2.7 spectrometer (9.6 GHz X band) with a rectangular TE102 microwave cavity at room temperature. WIN EPR software (Bruker) was used to analyze the data. Native DEP spectra were collected for both the solid phase and as an aqueous suspension in 100 mM phosphate buffer. Particles after organic or acid extraction were suspended with 100 mM phosphate buffer. Spectra of the organic extracts of DEP were also recorded. Instrument parameters were as follows: Microwave frequency, 9.788 GHz; microwave power, 10 mW; receiver gain, 1.00e+005; modulation frequency, 100 KHz; modulation amplitude, 4.00 G; time constant, 20.480 ms; sweep time, 41.943; sweep width, 500 G, and center field, 3491.67 G. Each spectrum is the result of three scans.
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RESULTS |
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DISCUSSION |
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![]() | (Reaction 1) |
![]() | (Reaction 2) |
Interestingly, the water soluble Vit. E analog, Trolox, and GSH showed the least activity while ascorbate and DTT displayed the highest (Fig. 1). The differences in the reactivity of these species are at this time difficult to rationalize using, for example, reduction potentials since the intimate details of the reactions are not known (i.e., outer-sphere versus inner-sphere electron transfers or nucleophilic addition-elimination chemistry). Moreover, major differences exist in the accessibility of these agents to possible reactive centers on DEP. Regardless, it is worth noting that ascorbate and vicinal thiols are particularly adept at performing the above described redox chemistry.
Importantly, the redox properties of DEP appear to be intrinsic as the majority of the catalytic properties remain even after multiple extractions with methylene chloride or aqueous acid. The intrinsic redox properties of the DEP are not necessarily covalently associated with the particle but rather, are considered to be inherent since they are not readily extracted under conditions which will be far more stringent than what can occur biologically. The idea that the toxicity of DEP can be due to the particles themselves as well as to the extractable components was demonstrated recently when Yanagisawa and coworkers (2003) reported that the DEP core, rather than organic extractables, was primarily responsible for the aggravation of LPS-mediated lung injury. From a toxicological perspective, this chemistry of DEP can be deleterious for several reasons. Depletion of intracellular reducing equivalents can change the redox status of a cell. It has been proposed that changes in cellular redox status towards greater oxidation can initiate cell signaling machinery leading to apoptosis and/or necrosis (Schafer and Buettner, 2001
). The catalytic nature of the DEP chemistry and the ability to generate ROS would lead to a prediction that they can be extremely proficient in altering intracellular redox status. Myriad studies have established the toxicity associated with excessive ROS generation (for one of many treatments of this topic, see Halliwell and Gutteridge, 1999
). In this study, we find that DEP are capable of catalyzing DNA damage in the presence of a reductant, including DTT or ascorbate.
It was found that DEP contain a stable and prevalent paramagnetic species. This finding is not surprising since other researchers have reported EPR signals indicative of paramagnetic organic species in cigarette tar and extracts (Stone et al., 1995; Zang et al., 1995
), airborne particles (Dellinger et al., 2001
), and even in C60 fullerene preparations (Paul et al., 2002
). The EPR signal in DEP is similar to those previously reported for semiquinone radical species in PM2.5 (mean aerodynamic diameter <2.5 microns) particulate matter (Dellinger et al., 2001
) and cigarette tar extracts (Stone et al., 1995
). It should not be surprising that similar signals are detected in PM2.5 since they likely include DEP (whose average size, 0.10.3 nM, would indicate their presence in the PM2.5 fraction). Since semiquinone radical species have been implicated in the generation of ROS in biological systems via redox cycling (Stone et al., 1995
), it may well be that a non-dissociable semiquinone radical species associated with the DEP is at least partially responsible for the generation of ROS by the reductants tested in this study.
This work begins to provide definition and characterization of the inherent chemistry of DEP. Recent studies indicating intracellular localization of DEP, and ultrafine PM, underscore the importance of establishing the toxicologically relevant chemistry of these particles. The results of this study indicate that DEP are themselves reactive entities which can catalyze the reduction of O2 by a variety of reducing agents, including biologically relevant reductants. This reactivity appears to be an intrinsic part of the particles since methylene chloride or aqueous acid extraction of DEP did not significantly alter their reactivity. Evidence for oxidative damage to DNA in the presence of DEP has also been observed. Finally, EPR analysis of the particles indicates that they contain paramagnetic species which are likely to be semiquinones, and which may participate in the redox processes. To be sure, it will be difficult to extrapolate the conditions of these chemical studies to actual in vivo DEP exposure. However, the results of this study indicate the possibility of particle-dependent chemical processes that can contribute, along with biologically extractable components, to the overall toxicity of DEP.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (310) 825-6267. E-mail: jfukuto{at}mednet.ucla.edu.
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