2004 Progress Report: The Chemical Toxicology of Particulate Matter

EPA 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 , Kumagai, Yoshito , Miguel, Antonio , Nel, Andre E. , Sioutas, Constantinos
Current Investigators: Cho, Arthur K. , Froines, John R.
Institution: University of California - Los Angeles , University of Southern California , University of Tsukuba
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, 2003 through May 31, 2004
RFA: Airborne Particulate Matter (PM) Centers (1999) RFA Text |  Recipients Lists
Research Category: Air Quality and Air Toxics , Particulate Matter , Air

Objective:

The objectives of this research are to use PM concentrators with a biosampler to collect coarse, fine and ultrafine PM from freeways and tunnels for in vitro chemical and biological studies and to characterize the chemical reactivity of the PM samples. The subaims are to measure the redox activity of PM from freeways and tunnels with heavy diesel traffic and heavy gasoline traffic, to determine concentrations of targeted chemical species based on their known reactivities and potential toxicity, and to apply newly established in vitro assays to measure electrophilicity or covalent binding to biomolecules to distinguish this reactivity from that involving the generation of reactive oxygen species.

Progress Summary:

1. Biosampler Studies: Redox Activity

a. Freeway Study. Biosampler based ambient PM samples were collected in the summer and winter at sites adjacent to a major gasoline vehicle freeway (110) in the Los Angeles Basin. All samples were assayed for redox activity using DTT as the electron source (see Table 1) (Cho, et al., 2005). The winter samples were subjected to a second assay using ascorbate as the electron source. Control experiments suggest that the DTT based activity is limited to redox active organic compounds whereas Mudway and his associates described an ascorbate based assay (Mudway, 2004) that was sensitive to metals as well as organics.

In addition to ascorbate as the electron source, we have added salicylate to the reaction media to monitor the metal based Fenton reaction by determining conversion of salicylate to dihydroxybenzoic acids (DHBA) (Themann, et al., 2001). The consumption of the antioxidants, DTT and ascorbate, were measured as a function of time and mass. Dihydroxybenzoate formation was determined in the presence of ascorbate and salicylic acid. The results were obtained from a single biosampler sample collected in the summer of 2004 and the winter of 2005.

The results are presented in Table 1. Overall exposure to redox active material is shown in the last column and represents the product of DTT activity per microgram and the PM concentration per m3. We found that the ascorbate based redox activity was higher than that due to DTT, in part due to the difference in conditions but also due to the presence of metals since much of the activity is blocked by the addition of a metal chelator. DHBA levels were highest in the UF fraction, a somewhat unexpected observation since metal content is lower in the UF fraction. However, the higher DTT activity of the UF fraction indicates a greater hydrogen peroxide generating capacity due to its organic constituents and DHBA formation may be reflective of hydrogen peroxide levels rather than metal content. The results are expressed per microgram of sample and the total exposure based on DTT redox activity is shown in the last column. Because of the day to day variability in ambient samples, it is difficult to draw conclusions about seasonal differences. However, there appears to be a trend toward greater redox activity in the summer months compared to the winter.

Table 1. Redox Properties of PM on the 110 Freeway

Table 1. Redox Properties of PM on the 110 Freeway

b. Caldecott Tunnel Study. The Caldecott Tunnel provided access to an enclosed environment of vehicular traffic. The tunnel has two bores, one of which is used by gasoline and diesel vehicles (CB1, Mixed) and a second which is used exclusively by gasoline vehicles (CB2, diesel). Again, samples were collected in the summer and winter seasons and assayed for redox activities. (Table 2). Subsequent to the collections the glass containers used for the winter samples were found to produce a redox artifact of ascorbate consumption. Accordingly, the ascorbate derived values for the two seasons cannot be directly compared. The DTT based activities were not affected by the glass components. The DTT redox activity observed suggests that the summer samples had greater activity than the winter samples, with the coarse fraction having the lowest activity.

Table 2. Redox Properties of PM From the Caldecott Tunnel

Table 2. Redox Properties of PM From the Caldecott Tunnel

2. Quinone Concentrations in Particle and Volatile Fractions

a. Caldecott Tunnel. In parallel collections to those with the biosampler, filter and volatile air samples were collected on Teflon filters (PM2.5) with XAD resin beds below the filters. The filters and XAD beds were extracted with dichloromethane and four quinones determined by GC/MS (Cho, et al., 2004) (Figure 1). The results from this analysis show that all four of the quinones were present with high levels of 9,10-anthraquinine (9,10-AQ) in the PM2.5 fraction and high levels of 1,4-NQ in the volatile, XAD, fraction. No clear seasonal pattern was apparent. The substantial day to day variability is shown by the standard deviations observed for 9,10-AQ in particular. This variability limits the ability to evaluate differences in levels for their significance.

Figure 1. Quinone Concentrations in the Caldecott Tunnel

Figure 1. Quinone Concentrations in the Caldecott Tunnel

Collections were made on four separate days and the filters and XAD resin media from extracted with dichloromethane. The extracts were assayed for the quinones shown above by GC/MS. The results are shown with standard deviations for at least three values except the winter CB1 collections for which only two samples were available.

b. Study of 5 Sites Along the Wind Trajectory in the Summer of 2005. In a study examining changes in air mass content, collections were made in five sites along the wind trajectory of the LA basin on each of four days. The collections were made with a Tisch sampler, which was sequentially moved along the trajectory on each day using Teflon filters and a XAD resin bed for the volatile fraction. The standard deviations demonstrate the day to day variability in the concentrations and emphasize the need for careful and consistent collection. The study focused on changes in the atmosphere as the air mass moved from the west end of the LA basin (Santa Monica) to the east, sequentially in Long Beach, Anaheim, Mira Loma and Riverside on each of four days. The model considered is that Santa Monica is primarily a source site, in which direct emissions from vehicular traffic are introduced into the air mass. The concentrations of the four quinones in the filter (PM2.5) and the volatile (XAD) fractions are shown in Figure 2.

Figure 2. Quinones in the LA Basin

Figure 2. Quinones in the LA Basin

These results, together with the results from the Caldecott Tunnel, are consistent with the following chemical transformations:

  1. 9,10-AQ is present as direct exhaust (Figure 1) and does not appear to increase as the air mass moves east, so it may not be a major product of photochemical transformations.
  2. The 9,10-PQ present in the ambient PM2.5 increases as the air mass moves east, suggesting (Figure 2) that it is being formed by photochemical reactions. Levels of its precursor, phenanthrene, were highest in Santa Monica, consistent with its role as a source site, and decrease as the air mass moves east and is diluted (data not shown).
  3. Similar arguments apply to 1,4-NQ which is found as direct exhaust (Figure 1) but also increases in the volatile fraction as the air mass moves east (Figure 2).
  4. 1,2-NQ levels do not appear to change with easterly movement (Figure 2) of the air mass, but its consistent presence in both tunnel and ambient air samples is consistent with its formation from exhaust and rapid decomposition. This is a reactive quinone.
  5. 9,10-Anthraquinone does not exhibit redox, electrophilicity or toxicity under the conditions tested, but may serve as a marker for vehicular traffic. It is present in all samples and does not follow the photochemical increase associated with 1,4­-NQ and 9,10-PQ.

The wide variability between days at the same location, could be due to differences in traffic density and possibly fuel sources, but the trends of the mean values are consistent with various hypotheses regarding generation of reactive compounds by the source and by photochemical changes at a receptor, i.e., low levels of photochemical products at sources and higher levels at receptors. The increase in quinone concentrations observed as the air mass proceeds to the receptor sites, suggests that adverse health effects associated with this class of reactive organic compound will also increase.

3. Electrophilic Properties

The electrophilic properties of PM samples are complementary to their redox properties. This property is associated with covalent bond formation with critical thiol functions and can result in a long lasting cellular insult. The problem with measuring electrophilicity has been one of sensitivity. To increase the signal resulting from a covalent interaction, we have assessed the use of thiol enzymes as targets for electrophiles, utilizing the amplification available from the catalytic and time dependent nature of the response. Thus, by catalyzing the formation of a new product with time, monitoring product formation over time will permit assessment of changes in catalytic activity with very low concentrations of inactivating agents.

a. Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH). This enzyme was found to be a key target of reactive quinones in a study using yeast as the target species {Rodriguez, 2004 #572}. Subsequent studies on the mechanism of its inhibition revealed that two mechanisms, one dependent on reactive oxygen species (ROS) and a second, oxygen independent mechanism involving covalent interactions with the catalytic thiol (Rodriguez, et al., 2005). The redox active 9,10-PQ inactivated the enzyme by both oxygen dependent and independent mechanisms whereas 1,4-benzoquinone (1,4-BQ), a pure electrophile in biological systems, inactivated the enzyme by a covalent, oxygen independent mechanism. The inactivation is characterized by two parameters, a rate constant for inactivation (k(inactivation)), and an affinity parameter, Ki, which reflects the concentration of inhibitor needed to decrease the overall rate by 50% under a standard set of conditions.

The rate constants for the two quinones, determined under anaerobic conditions are shown in table 3, together with the k(inactivation) for a diesel exhaust extract that we have used in all of our experiments as a “standard”. Experiments with diesel exhaust particle (DEP) extracts showed an analogous time dependent inactivation that could be prevented by coincubation with high concentrations of alternate thiols, consistent with covalent attachment to the enzyme thiol. These results are the first to demonstrate the electrophilic nature of DEP constituents and indicate that with this source of PM, the induction of cellular stress by pathways other than reactive oxygen generation is possible.

Table 3. GAPDH Inactivation

Table 3. GAPDH Inactivation

b. Protein Tyrosine Phosphatase (PTP). PTPs are a class of phosphatases that participate in the regulation of a variety of kinases, including those associated with tyrosine kinase based receptors such as the epidermal growth factor receptor (EGFR). This receptor system is involved in cell proliferation and recently has been implicated in asthma pathophysiology, specifically with respect to airway remodeling and mucin secretion (Hamilton, et al., 2005). The receptor system is activated by ROS through the inactivation of PTP 1B after which it rapidly recovers. However, when exposed to 1,2-NQ, the enzyme is inactivated and studies with bronchial smooth muscle preparations have shown that the receptor is activated. Colleagues at Tsukuba University (Kikuno, et al., 2005) demonstrated that 1,2-NQ forms covalent bonds with protein tyrosine phosphatase 1B. The covalent nature of this interaction suggests that these effects would be long lasting and could contribute to a chronic state of restricted airway passage. This enzyme could also be used as a probe for electrophilic properties of PM samples and would be particularly relevant because of its involvement of EGFR in the pathophysiology of asthma. Studies developing this target are planned.

4. Cellular Toxicity

a. Yeast. Yeast (Saccharomyces ceriviseae) cells have the ability to grow in the presence and absence of oxygen so that the role of oxygen in toxicity of test substances can be readily assessed (Rodriguez, 2004 #572). For example, 1,4-benzoquinone, whose action is based on its electrophilicity or covalent bond forming action, is equipotent in the presence or absence of oxygen whereas 9,10-PQ exhibited greater toxicity in the presence of oxygen (Rodriguez, 2004 #572). The toxicity data are quantitated by the estimation of an EC50 or the concentration needed to reduce cell viability by 50%.

Studies with DEP extract showed oxygen dependent and independent toxicity based on growth inhibition and cell viability during the exponential growth phase that was proportional to extract concentration. Under both conditions, levels of the intracellular antioxidant, glutathione (GSH), were reduced in proportion to the loss of cell viability consistent with the notion of oxidative stress. The ability of DEP extract to induce a state of oxidative stress in the absence of oxygen is again consistent with the observations with GAPDH that indicate an electrophilic component in the extract.

b. RAW 247.6 Cells. This murine macrophage cell line has been used in numerous investigations of PM toxicity (e.g., Hiura, et al., 1999; Li, et al., 2000; Li, et al., 2002; Chin, et al., 2003; Li, et al., 2004). In our initial studies, we examined the actions of various toxins and PM samples on cell viability, measured by ATP depletion. Whenever possible, dose response data were collected to determine the EC50 or concentration needed to deplete ATP levels by 50% of control. An EC50 value of 60 μg/mL has been consistently observed for DEP extracts. Two ambient PM samples, a filter sample collected at the USC site and the winter samples from the tunnel have been evaluated by this assay. Preliminary results are shown in Table 4.

Table 4. Redox Activity and Cellular Toxicity of PM Samples

Table 4. Redox Activity and Cellular Toxicity of PM Samples

DTT based redox activities, shown for the samples evaluated, did not correlate with toxicity. The toxicity of the tunnel samples was much lower than that of DEP or filter extracts and 50% ATP depletion was not observed even after a high concentration of the PM sample. ATP depletion is a non specific marker for cellular toxicity and may not be sufficiently sensitive for PM samples. Accordingly, we are assessing alternate probes for cellular responses.

5. Cellular Oxidative Stress as Determined by Heme Oxygenase-1 (HO-1) Expression

Bore 1 & 2 samples collected in the Caldecott Tunnel were compared for the ability of coarse and ultrafine particles to induce HO-1 expression. We have previously demonstrated that HO-1 is a sensitive oxidative stress marker that is induced by ambient PM through their ability to release the Nrf2 transcription factor to the nucleus of epithelial cells and macrophages. In the comparative study in RAW 264.7 cells, we showed that all the particles were able to induce HO-1 production. However, the ultrafine particles were somewhat more potent compared to the larger particles, which is in agreement with what we have previously seen in ambient air collected in downtown Los Angeles. In the experiment shown, Bore 2 (LDV) particles showed increased ability to induce HO-1 compared to Bore 1 (HDV, LDV) samples but this was not uniformly seen in all our experiments.

Caldecott particles induce HO-1 production.

Future Activities:

Work to be Completed Before May 31, 2006

  1. Samples from the 710 freeway will be assayed for relevant redox activity.
  2. Samples from a receptor site in the East San Gabriel Valley will be subjected to redox assays.
  3. Assessment of the GAPDH based electrophile assay will be completed and it will be applied to an ambient PM sample.
  4. The assay utilizing salicylate oxidation to determine metal based redox activity will be assessed for its suitability in PM characterization.
  5. The effects of prooxidants and electrophiles on cellular glutathione and its disulfide will be examined as an alternative to ATP depletion.
  6. Analysis will be conducted to compare the results of the Caldecott and the 110 gasoline freeway studies

References:

Chin BY, Trush MA, Choi AM, Risby TH. Transcriptional regulation of the HO-1 gene in cultured macrophages exposed to model airborne particulate matter. American Journal of Physiology-Lung Cellular and Molecular Physiology 2003;284:L473-80.

Cho A, Di Stefano E, Ying Y, Rodriguez CE, Schmitz DA, Kumagai Y, Miguel AH, Eiguren-Fernandez AT, Kobayashi TE, Avol E, Froines JR. Determination of four quinones in diesel exhaust particles, SRM 1649a and Atmospheric PM2.5. Aerosol Science and Technology 2004;38:68-81.

Cho AK, Sioutas C, Miguel AH, Kumagai Y, Schmitz DA, Singh M, Eiguren-Fernandez A, Froines JR. Redox activity of airborne particulate matter at different sites in the Los Angeles Basin. Environmental Research 2005;99:40-47.

Hamilton LM, Puddicombe SM, Dearman RJ, Kimber I, Sandstrom T, Wallin A, Howarth PH, Holgate ST, Wilson SJ, Davies DE. Altered protein tyrosine phosphorylation in asthmatic bronchial epithelium. European Respiratory Journal 2005;25:978-85.

Hiura TS, Kaszubowski MP, Li N, Nel AE. Chemicals in diesel exhaust particles generate reactive oxygen radicals and induce apoptosis in macrophages. The Journal of Immunology 1999;163:5582-91.

Kikuno S, Taguchi K, Iwamoto N, Yamano S, Cho AK, Froines JR, Kumagai Y. 1,2-Naphthoquinone activates vanilloid receptor 1 through increased protein tyrosine phosphorylation, leading to contraction of guinea pig trachea. Toxicology and Applied Pharmacology 2005.

Li N, Kim S, Wang M, Froines J, Sioutas C, Nel A. Use of a stratified oxidative stress model to study the biological effects of ambient concentrated and diesel exhaust particulate matter. Inhalation Toxicology 2002;14:459-86.

Li N, Venkatesan MI, Miguel A, Kaplan R, Gujuluva C, Alam J, Nel A. Induction of heme oxygenase-1 expression in macrophages by diesel exhaust particle chemicals and quinones via the antioxidant-responsive element. The Journal of Immunology 2000;165:3393-401.

Li N, Alam J, Venkatesan MI, Eiguren-Fernandez A, Schmitz D, Di Stefano E, Slaughter N, Killeen E, Wang X, Huang A, Wang M, Miguel AH, Cho A, Sioutas C, Nel AE. Nrf2 is a key transcription factor that regulates antioxidant defense in macrophages and epithelial cells: protecting against the proinflammatory and oxidizing effects of diesel exhaust chemicals. The Journal of Immunology 2004;173:3467-81.

Rodriguez CE, Fukuto JM, Taguchi K, Froines J, Cho AK. The interactions of 9,10-phenanthrenequinone with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a potential site for toxic actions. Chemico-Biological Interactions 2005;155:97-110.

Themann C, Teismann P, Kuschinsky K, Ferger B. Comparison of two independent aromatic hydroxylation assays in combination with intracerebral microdialysis to determine hydroxyl free radicals. Journal of Neuroscience Methods 2001;108:57-64.

Journal Articles:

No journal articles submitted with this report: View all 10 publications for this subproject

Supplemental Keywords:

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

Relevant Websites:

http://www.scpcs.ucla.edu Exit

Progress and Final Reports:

Original Abstract
  • 1999
  • 2000
  • 2001 Progress Report
  • 2002 Progress Report
  • 2003 Progress Report
  • Final Report

  • 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)