2003 Progress Report: The Chemical Toxicology of Particulate MatterEPA Grant Number: R827352C001
Subproject: this is subproject number 001 , established and managed by the Center Director under grant R827352
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Southern California Particle Center and Supersite
Center Director: Froines, John R.
Title: The Chemical Toxicology of Particulate Matter
Investigators: Cho, Arthur K. , Froines, John R. , Fukuto, Jon
Current Investigators: Cho, Arthur K. , Froines, John R.
Institution: University of California - Los Angeles
EPA Project Officer: Chung, Serena
Project Period: June 1, 1999 through May 31, 2005 (Extended to May 31, 2006)
Project Period Covered by this Report: June 1, 2002 through May 31, 2003
RFA: Airborne Particulate Matter (PM) Centers (1999) RFA Text | Recipients Lists
Research Category: Air Quality and Air Toxics , Particulate Matter , Air
The objective of this research project is to focus on the central hypothesis of the Southern California Particle Center and Supersite (SCPCS), which is that organic constituents associated with particulate matter (PM)—including quinones, other organic compounds (polycyclic aromatic hydrocarbons [PAHs], nitro-PAHs, and aldehydes/ketones), and metals—are capable of generating reactive oxygen species and acting as electrophilic agents. They have a central role in allergic airway disease such as asthma and cardiovascular effects through their ability to generate oxidative stress, inflammation, and immunomodulating effects in the lungs and airways.
This is one of the project progress reports for the SCPCS. The progress for the other research projects conducted by the Center is reported separately (see reports for R827352, R827352C001 through R827352C006, and R827352C008 through R827352C021).
This project is testing the hypothesis that PM contains reactive chemical species and that they, either separately or as a mixture, are responsible for the toxicological phenomenon associated with PM. These chemical species can be organic or inorganic and act through several possible chemical reactions with biological substrates. We are focusing on two general mechanisms: redox and electrophilic reactions. Redox reactions involve the catalytic reduction of oxygen to reactive oxygen species by components of PM with electrons from biological sources. In the electrophilic reactions, a reactive function in PM reacts with nucleophilic functions in biological systems to form covalent bonds. These are irreversible so the biological molecule affected is destroyed. By characterizing and quantitatively determining the reactivity in a given PM sample, we hope to be able to predict its toxicity. The thiol function on biological macromolecules is a likely target of these reactions as they react in both redox and covalent bond-forming reactions. Thiols are key functions in biological molecules such as enzymes, transporters, and receptors so their modification will result in substantial disruption of cell biochemistry.
To test our hypotheses, we have developed and continue to develop quantitative chemical assay procedures that can be applied to PM samples to assess their chemical reactivity and toxicity as predicted by the considerations above. Two of the assays, an assay for selected quinones and an assay for redox activity, have been developed and applied to ambient samples. Additional assays under development include an electrophilic reactivity assay and two biochemical assays.
Aim 1: Dithiothreitol (DTT)-Based Redox Assay
We have analyzed Los Angeles Basin PM samples from several SCPCS projects to determine the relationships between source and distance, meteorology changes, and redox activity in samples collected from various sites. We have made the following observations:
- Redox activity is greatest on a mass basis in ultrafine fractions and correlates with the organic and carbon content of the particles rather than the transition metal content.
- The assay appears to be reproducible in other laboratories (a comparison of results obtained in this laboratory and those obtained by a Mexico City group on the same samples indicates good agreement).
- Initial results of a study comparing our DTT-based assay with that based on ascorbate used by Professor Kelly of London suggest that an ascorbate-based assay may be measuring metal-based redox activity, whereas our results with DTT appear to correlate with organic content. We now are investigating the differences in the two reactions by determining the consumption of oxygen as well as the reducing agents to obtain the stoichiometry of the reaction.
- In studies directed at the chemical characterization of PM, we examined the effect of extraction of diesel exhaust particles (DEPs) on the residual redox activity. We found that extraction of DEPs with dichloromethane had only a slight effect on the redox activity of the particles, but the dichloromethane extract had about 50 percent of the activity found in the original DEP sample. These results suggest that the chemical properties of particle samples change after extraction, and we now are testing this notion with ambient PM samples collected on filters.
Aim 2: Electrophile Assay
To address the second pathway for oxidative stress induction by PM, we decided to use an enzyme with a key thiol that, when modified by covalent interaction, would lose activity. Because of its catalytic nature, enzyme activity can be amplified with a sensitive product assay and longer incubation. In preliminary studies with a common enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), we have found that electrophilic quinones as well as diesel exhaust extracts irreversibly inhibit this enzyme under anaerobic conditions. DEP extracts will inactivate the enzyme at concentrations of 10 to 100 μg/mL. Because of the small volumes used in the procedure, the assay can be applied to filter or impinger PM samples collected at sites proximal to freeways.
Aim 3: Yeast-Based Toxicity
Both redox and electrophilic activity cause oxidative stress to cells, but they differ in their dependency on oxygen (Rodriguez, et al., in press, 2004). We began studies with yeast to test the hypothesis that the two mechanisms of oxidative stress could be distinguished with this system because these cells grow under both aerobic and anaerobic conditions. In experiments designed to test ambient particle samples, yeast cells were exposed to organic extracts of DEPs at concentrations of 10 to 100 μg/mL. The cell viability of yeast was inhibited by 50 percent at about 25 μg/mL concentrations. Removal of oxygen reduced the DEP extract toxicity threefold, indicating that most, but not all, of the toxicity was oxygen dependent. Consistent with the notion of oxidative stress, the intracellular concentration of glutathione, which maintains the redox status of the cell, was decreased by 40 percent at a concentration of 10 μg/mL. Although this concentration was not lethal, it clearly subjected the cell to oxidative stress.
Aim 4: Study of Intact Particles Versus Extracts
Numerous studies have shown that PM can be toxic as intact particles, and studies examining their inherent chemical activity have been conducted. In the initial study, we examined DEPs using a modification of the DTT assay in which we measured oxygen consumption instead of DTT consumption in an enclosed system. We found that as much as 90 percent of the activity found in the original particles remained after three extractions with dichloromethane. To test the notion that the residual activity may be a result of adsorbed metals, the particles then were extracted with dilute acid (1 N HCl). Again, about 70 percent of the activity remained in the particles (Pan, et al., in press, 2004). These results indicate that particles such as those from diesel exhaust exhibit a reactivity that is not removed by the common extraction procedures for PM studies. Furthermore, as the sum of the extracted activity and the residual particle activity is greater than the original particle activity, dichloromethane extraction appears to alter the redox properties of the residual particle. These studies will continue with filter-collected particles obtained from sites on the University of Southern California campus.
In Year 6 of the project, we will continue to develop quantitative assay procedures that can be applied to PM samples to assess their chemical reactivity and toxicity as predicted by the considerations above. In particular, we will complete the development of the GAPDH assay and assess the application of assays to Center projects examining health effects of ambient particles. The purpose of the assay procedures is to characterize the chemical properties of PM with the following objectives: (1) to determine the comparative toxicities of PM at emission sources and at receptor sites in winter and summer; and (2) to determine the comparative toxicities of emissions from gasoline and diesel fuel sources.
Journal Articles:No journal articles submitted with this report: View all 10 publications for this subproject
Supplemental Keywords:particulate matter, PM, quinones, polycyclic aromatic hydrocarbons, PAHs, aldehydes, ketones, metals, allergic airway disease, human exposure studies, asthma, cardiovascular effects, aerosol sampling, atmospheric aerosol, environmental monitoring, California, CA, acute exposure, aerosols, air pollution, air quality, air toxics, airway disease, allergen, allergic response, ambient aerosol, assessment of exposure, asthma triggers, atmospheric chemistry, bioaerosols, biological response, environmental hazard exposures, environmental health hazard, environmental triggers, environmentally caused disease, epidemiology, exposure assessment, health effects, human exposure, human health effects, inhaled particles, lead, particle transport, particulate exposure, particulates, sensitive populations, toxicology, toxics,, RFA, Health, Scientific Discipline, Air, HUMAN HEALTH, particulate matter, Environmental Chemistry, Air Pollutants, Risk Assessments, Biochemistry, Health Effects, Biology, particulates, ambient aerosol, asthma, toxicology, quinones, human health effects, airway disease, allergic airway disease, air pollution, PAH, particulate exposure, human exposure, toxicity, breath samples, allergens, particle concentrator, airborne urban contaminants, human health risk, genetic susceptibility, aerosols, atmospheric chemistry, dosimetry
Progress and Final Reports:Original Abstract
Main Center Abstract and Reports:R827352 Southern California Particle Center and Supersite
Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R827352C001 The Chemical Toxicology of Particulate Matter
R827352C002 Pro-inflammatory and the Pro-oxidative Effects of Diesel Exhaust Particulate in Vivo and in Vitro
R827352C003 Measurement of the “Effective” Surface Area of Ultrafine and Accumulation Mode PM (Pilot Project)
R827352C004 Effect of Exposure to Freeways with Heavy Diesel Traffic and Gasoline Traffic on Asthma Mouse Model
R827352C005 Effects of Exposure to Fine and Ultrafine Concentrated Ambient Particles near a Heavily Trafficked Freeway in Geriatric Rats (Pilot Project)
R827352C006 Relationship Between Ultrafine Particle Size Distribution and Distance From Highways
R827352C007 Exposure to Vehicular Pollutants and Respiratory Health
R827352C008 Traffic Density and Human Reproductive Health
R827352C009 The Role of Quinones, Aldehydes, Polycyclic Aromatic Hydrocarbons, and other Atmospheric Transformation Products on Chronic Health Effects in Children
R827352C010 Novel Method for Measurement of Acrolein in Aerosols
R827352C011 Off-Line Sampling of Exhaled Nitric Oxide in Respiratory Health Surveys
R827352C012 Controlled Human Exposure Studies with Concentrated PM
R827352C013 Particle Size Distributions of Polycyclic Aromatic Hydrocarbons in the LAB
R827352C014 Physical and Chemical Characteristics of PM in the LAB (Source Receptor Study)
R827352C015 Exposure Assessment and Airshed Modeling Applications in Support of SCPC and CHS Projects
R827352C016 Particle Dosimetry
R827352C017 Conduct Research and Monitoring That Contributes to a Better Understanding of the Measurement, Sources, Size Distribution, Chemical Composition, Physical State, Spatial and Temporal Variability, and Health Effects of Suspended PM in the Los Angeles Basin (LAB)