2002 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 , Michigan State University , University of California - Irvine , University of Southern California
Current 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, 2001 through May 31, 2002
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, 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.
The hypothesis explored during Year 4 of the project is that certain organic functional groups and transition metals present in PM are capable of interacting with cellular systems and molecules to generate reactive intermediates that alter thiol functions in cells. These alterations could be either changes in redox state or covalent alterations that result in signaling changes and the precipitation of inflammatory or other toxic responses. The project is determining the redox activity of PM fractions, the concentration of quinones, a surrogate of the reactive species present in PM, and is developing probes for assessing electrophile-based toxicity. In addition, tissue studies are being conducted to develop a cellular toxicity assay that reflects the chemical interactions described above. Currently, we are focusing on the capsaicin receptor.
Gas Chromatography/Mass Spectrometry (GC/MS) Analysis of Selected Quinones. The GC/MS assay of the four quinones—1,2- and 1,4-naphthoquinone; 9,10-phenanthroquinone; and 9,10-anthraquinone—together with data on ambient PM, has been accepted for publication by Aerosol Science and Technology. Quinone levels in filter samples from the Children's Health Study (CHS) sites have been determined, but we want to determine total quinones in both particle and volatile phases. High levels of the naphthoquinones (50 X that on filters) were found in this volatile fraction. These data provide a basis for estimating exposure in different Los Angeles Basin (LAB) sites.
The Dithiothreitol (DTT)-Based Redox Assay. Samples of PM have been collected at sites used in the freeway and source-receptor studies with a mobile concentrator for coarse, fine/ultrafine, and ultrafine particle fractions. The samples were subjected to in vitro-based toxicity and DTT-based redox assays. The multiple analyses performed in this project have permitted several conclusions:
- The ability to induce oxidative stress, evidenced by hemeoxygenase-I induction, is highest with the ultrafine component, with the ultrafine/fine mixture next, and with the coarse particles least.
- Consistent with the notion of redox-based oxidative stress, the DTT assay also was highest in the ultrafine fraction, and the correlation between DTT activity and hemeoxygenase induction activity of PM was excellent.
- PM from the University of Southern California (USC) site, which is upwind from freeways, was the least active. Claremont, a site in eastern LAB, had the highest activity.
- The difference in DTT-based redox activity between two freeway sites (50 m and 150 m from the center line) was not significantly different.
Electrophile Assay. The electrophile assay is based on the notion that a covalent bond could be formed between reactive functional groups such as a 1,3-ene-one system or Michael acceptors. We have synthesized a thiol derivative of dansyl acid that will react with Michael acceptors to form irreversible adducts for quinones. We have synthesized test adducts, including naphthoquinone and benzoquinone, and are optimizing reaction conditions for other substrates.
Capsaicin Receptor Assay. Our colleagues in Tsukuba, Japan, have shown that 9,10-phenanthroquinone causes a concentration-dependent contraction of guinea pig tracheal rings that is blocked by the antioxidant enzyme superoxide dismutase (SOD). In contrast, 1,2-naphthoquinone causes a concentration-dependent contraction of this tissue, but by a mechanism that is SOD insensitive. To build on this work, we are developing an assay for the capsaicin receptor system that is based on a bronchial airway cell line, BEAS-2B, which responds to capsaicin agonists with an increase in calcium ion influx (Veronesi, et al., 1999; Oortgiesen, et al., 2000). Using this cell line, we will determine further details of the capsaicin activation process with two aims to: (1) determine the mechanism by which the quinone probe activates, and (2) develop a sensitive assay that could be used in toxicity determinations of ambient PM.
Biochemical Assays. Yeast cells are being evaluated as a means of rapid redox-based toxicity. Preliminary results indicate that yeast cells are resistant to redox-based toxins, and we are studying strains that are devoid of glutathione synthesis enzymes. Glutathione will protect the cell against redox and electrophilic toxins, and its removal will enhance the sensitivity of yeast to these toxins.
Work With Dr. Jon M. Fukuto. The hypothesis being tested is that diesel exhaust particles (DEPs) are self-contained, catalytic entities that are capable of performing potentially toxic chemistry when in the proximity of metabolizing/respiring cells. We have observed that DEPs are capable of acting as catalysts for the reduction of dioxygen by a variety of biological reducing agents (nicotinamide-adenine dinucleotide, ascorbate, thiols). This indicates that DEPs can act as catalysts for the generation of reactive oxygen species in biological systems.
We will continue working on the following assays:
- Electrophile Assay—The reaction conditions between selected electrophiles will be determined to establish a general protocol that will measure electrophile activity in both aromatic and aliphatic compounds. The assay will be applied to PM samples from the freeway studies, which will be processed for DTT and quinones.
- Yeast-Based Toxicity Assay—We are attempting to increase the sensitivity of this assay by targeting specific thiol groups in yeast.
- Capsaicin Receptor Activation Assay—We will learn how to manipulate the BEAS-2B cells and assay their uptake of the calcium ion. Subsequently, a series of dose response curves to capsaicin agonists and quinones will be determined. Using the vanilloid receptor (VR) antagonist, capazepine, the relative activity on the VR will be determined, and the assay will be applied to selected PM samples.
- Fractionation of PM—PM will be extracted and fractionated initially by procedures such as those of Alsberg (Alsberg, et al., 1985) or Durant (Durant, et al., 1998). The polar fraction will be further separated by liquid chromatography.
- Electrochemical and Spectroscopic Studies of DEPs With Jon Fukuto—We will continue with our electrochemical (O2 uptake, CV), biological (DNA nicking), and spectroscopic (electron spin resonance) studies.
Application projects include the following:
- CHS—PM sample collection will continue at the CHS sites, with some of the volatile fraction samples being analyzed for quinones. When possible, these fractions will be analyzed for electrophilicity as well as DTT activity.
- Freeway Study—Additional sample collections are planned. We propose that collections at several sites less than 50 m from the freeway be analyzed for DTT activity, determining whether potential toxicity will decline with distance.
- Source/Receptor Study—This study will parallel the freeway study; additional samples will be collected at the USC and Riverside sites, but on the same day. These samples will be analyzed with DTT, quinone, and PAH assays.
- Joint Project With Other Centers—The Center agreed to begin an exchange study with Professor Kelly (Cambridge University, United Kingdom) who also has examined PM for redox activity by means of an antioxidant depletion assay. PM samples and assays will be exchanged and the results compared. Other analogous sample and assay exchanges have been proposed with Robert Devlin of the U.S. Environmental Protection Agency.
Journal Articles:No journal articles submitted with this report: View all 10 publications for this subproject
Supplemental Keywords:Particulate matter, quinones, PAHs, aldehydes, ketones, metals, allergic airway disease, human health risk, asthma, cardiovascular effects, oxidative stress, environmental monitoring, California, acrolein sampling, carbonyls, motor vehicle emissions, mobile sources, thiol function, redox activity, biochemical assays, diesel exhaust particles,, RFA, Health, Scientific Discipline, Air, HUMAN HEALTH, particulate matter, Environmental Chemistry, Air Pollutants, Risk Assessments, Biochemistry, Health Effects, Biology, ambient aerosol, asthma, particulates, human health effects, toxicology, quinones, airway disease, allergic airway disease, air pollution, PAH, human exposure, toxicity, particulate exposure, allergens, breath samples, aerosols, atmospheric chemistry, dosimetry, human health risk, genetic susceptibility, particle transport, particle concentrator
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)