2008 Progress Report: The Chemical Properties of PM and their Toxicological Implications

EPA Grant Number: R832413C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R832413
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Southern California Particle Center
Center Director: Froines, John R.
Title: The Chemical Properties of PM and their Toxicological Implications
Investigators: Cho, Arthur K. , Froines, John R. , Kumagai, Yoshito
Current Investigators: Cho, Arthur K. , Froines, John R. , Harkema, Jack , Fukuto, Jon , Kumagai, Yoshito
Institution: University of California - Los Angeles , University of Tsukuba
Current Institution: University of California - Los Angeles , Michigan State University , University of Tsukuba
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2012)
Project Period Covered by this Report: October 1, 2007 through September 30,2008
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Health Effects , Air

Objective:

The objective of the project is to characterize the catalytic redox and electrophilic properties of ambient PM samples using cell-free chemical assays.  The hypothesis being tested is that PM contain constituents that are capable of inducing cellular stress by redox or other chemical processes and that such processes can be quantitatively assessed by specific analytical chemical procedures.

 
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.  These reactions are thought to induce a state of oxidative stress in cells. In the electrophilic reactions, a reactive function in PM reacts with nucleophilic functions in biological systems to from covalent bonds.  These bonds are irreversible so that the affected biological molecule is destroyed.  The thiol group is a likely target of the two reactions as this group participates in both redox and covalent bond forming reactions.  Thiols serve key functions in proteins such as enzymes, transporters and receptors so their modification can result in substantial disruption of cell biochemistry.  By characterizing and quantitatively determining the reactivity in a given PM sample with respect to these chemical properties, we hope to be able to predict its potential toxicity. 
 
To date, we have developed three assay procedures that can be applied to PM samples to assess their chemical reactivity and potential toxicity as predicted by the considerations above.  The assays have been applied to different samples collected at sites in the Los Angeles Basin.  

Approach:

In the next phase of this research, we plan to apply the redox assay and two other chemical assays, one of which determines the reaction with thiols, to assess differences in chemical reactivity among major source types, season and size fraction in PM samples from the LAB. These differences will be analyzed in terms of the chemical constituents found in Project 1 and used to interpret the toxicological findings from Projects 2 and 4. We shall investigate the quantitative relationship between ROS chemistry and intracellular measures of oxidative stress and cellular toxicity. In a second component of the Project, we will study the interaction between carbon black particles and various adsorbed compounds to determine the effect of the particle matrix on chemical and biological activity. These studies are based on our observations that demonstrate residual redox activity in diesel exhaust particles after extractions and those of others showing differences in cellular toxicity of organic compounds when they are adsorbed onto particles. We will thus establish an experimental system of carbon black particles of varying dimensions to which selected organic and inorganic species are adsorbed. The chemical and biological properties of the particles will be determined and the effects of the matrix on the actions of the adsorbed species will be assessed.

Progress Summary:

Part of the role of the project 3 investigators is to provide data to other projects for their studies in which the assays developed are applied to samples.  In addition, project 3 continues to develop or refine the assays used to enable us to provide more detailed analyses.  In that context, we are collecting samples specifically for use as research objects.  In year 2 and 3, we obtained two sets of such samples. One set was obtained from the EPA and they were used to characterize the physical and chemical properties of the reactive substances found in the media.  A second set of samples was collected as part of Project 5 in Riverside, California to assess the relative contributions of particle and vapor phases to the chemical properties of toxicological interest. 
 
Chemical assays:
The chemical reactivity assays provide quantitative data on:
a.       Electron transfer capacity of organic and inorganic constituents, using the consumption of dithiothreitol (DTT) as the measure of activity.
b.      Electron transfer capacity of redox active metal constituents, using the conversion of salicylate to dihyroxybenzoic acids (DHBAs) by hydroxyl, a reaction dependent on transition metals.  A description of this assay and its application to ambient samples has been submitted for publication.
c.       The content of electrophilic species, capable of forming covalent bonds with a thiolate enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in PM samples.  The ability of the sample to inactivate the enzyme under anaerobic conditions is determined. 
d.      Specific chemical species relevant to the operating hypothesis are also measured, specifically:
i.        Polynuclear aromatic hydrocarbons
ii.      Selected quinones
iii.    Where necessary, metal ion species have been measured by inductively coupled plasma mass spectrometry through the analytical services of the Department of Chemistry at UCLA
e.       In a cellular assay, changes in oxidative status during exposure is determined.  The induction of the stress protein hemeoxygenase-1 and the changes in the redox status of glutathione are measured.
 
The accomplishments summarized in this report reflect the ongoing development and utilization of the assays to further characterize ambient air. 
 
Accomplishments 2005-06
The GC/MS based quinone assay and the DTT based redox activity assay were used in studies with DEP obtained from Japan and ambient air samples collected on the 110 freeway in Los Angeles and in the Caldecott Tunnel in the Berkeley Area for analysis.
 
Analyses of the four quinones in the Tunnel samples showed high concentrations of the naphthoquinones in the vapor fraction with relatively high levels of 9,10-anthroquinone in the particle phase.  The high levels of the naphthoquinones in the Tunnel were interpreted to indicate quinone formation during combustion rather than from photochemical processes. Redox analyses of freeway and tunnel samples revealed that, on a per mass basis, the activities were similar, although there was considerable day-to-day variation.  The difference between the Tunnel and freeway was in particle count; the count was much higher in the Tunnel so exposure was greater on a m3 basis.  Application of the GAPDH assay to DEPs showed, for the first time, that electrophiles are present in the organic extracts of these particles and similar findings were made in the ambient samples tested.  The assay is being refined in order to provide a robust and reproducible procedure that will provide quantitative data to allow comparison between samples.
 
Accomplishments 2006-07
In addition to analyses provided for samples collected in Project 1, work during this project period has continued to develop assay procedures for the ability of PM samples to carry out chemical reactions that reflect mechanisms of their toxicity.   We focused on two procedures, one an assay for metal based redox activity and the second, an assay for electrophile content.
 
Summary: Evaluation of the DHBA procedure for redox active metal species.
An assay for the electron transfer capacity of redox active metal constituents of air pollutants is being developed in which the conversion of salicylate to dihyroxybenzoic acids (DHBAs) by hydroxyl, is monitored. In study, assay results from a VACES-biosampler study of ultrafine particle suspensions, collected indoors and outdoors in the same site, were compared with copper and iron ion concentrations, obtained by IPCMS procedures.  The results indicated that copper ion was 60 times more active than iron in the Fenton reaction.  Therefore, the iron ion concentration would have to be at least 10 fold higher than that for copper in order to contribute significantly to the observed DHBA formation rate.  Thus, with this caveat, the assay can be used to assess metal based redox activity in a sample under conditions resembling those in lung lining fluid where high levels of ascorbate are present. A manuscript has been submitted for publication.   
 
The presence of electrophiles in PM samples
The thiol protein, GAPDH is used as a target nucleophile to determine the concentration of electrophilic species in a given test sample.  The reaction of the electrophile with the nucleophilic center in the enzyme is conducted under anaerobic conditions to prevent oxygen based inactivation.  The assay was used to measure electrophiles in extracts of diesel exhaust particles and in aqueous suspensions of ambient air samples collected in Riverside and Claremont, sites on the eastern end of the Los Angeles Basin. The electrophile content in DEP extracts were found to correlate with PAH and with quinone content, indicating that the electrophiles measured had similar physical chemical properties to these classes of organic compounds. Because of the irreversible nature of electrophile action on thiols, effects of chronic exposure to low levels of electrophiles could be cumulative, increasing the levels of protein thiol inactivation with time.  This action differs from the oxidation of thiols by ROS, which is reversible because cells can reduce oxidized thiols by processes involving disulfide isomerases and disulfide reductases2,4
 
Accomplishments 2007-08
In addition to the analysis of samples from project 1, additional studies utilized the assays developed to address the following questions:
1.      In the course of normal exposure, individuals are exposed to particle and vapor phase components of ambient air.  How are the reactive components of air, as defined by the assays, distributed between water and particles in suspension? 
2.      Does the vapor phase contain reactive compounds relevant to toxicity?  If so, what types of compounds and what kind of exposure is involved?
 
These questions were addressed with large scale samples, including DEP samples obtained from the EPA and samples from several large scale collections made in Riverside, CA. 
 
Study 1.  Characterization of 7 diesel exhaust particle (DEP) samples collected at the EPA
Initially, the physical properties of the reactive species were determined by filtering aqueous suspensions of the samples into particle (filter bound) and soluble (filtrate) fractions.  The activities of the two fractions were determined by assaying the total suspension and the filtrate, with the particle phase determined by difference.  Next, the activities of dichloromethane (DCM) extracts of the samples were compared to assess the polarity of the reactive species.  Finally, the DCM extracts were subjected to reductive acetylation to determine the role of quinone like species in the reactions.  Reductive acetylation is used in stabilizing quinones for a GC/MS assay1 and will block the redox and electrophilic reactions of quinones.
 
Results:  Experiments with aqueous suspensions of DEP showed that the chemical species associated with redox activity are bound to particles, whereas electrophiles are mostly water soluble.  However, this extract does not exhibit metal based redox activity and both the redox and most of the electrophilic activities are lost with reductive acetylation.  One possible explanation for the fractionation results is the association of humic like substances with the particles.  Humic like substances have been reported in diesel exhaust particles3 which are thought to be polymeric, highly polar organic materials5.  Included in the functional groups are quinones, catechols and multiple carboxyl and hydroxyl functions.  The latter two functions could act as chelating functions for metals and the first two functions could account for the electrophilic and redox activities found in the particles.  Reductive acetylation of the DCM extracted provided support for the involvement of quinone-like organic species in both redox and electrophilic activities of this fraction. 
 
Study 2: Large scale sample collection at Riverside.  Tisch sampler with filter and XAD resin.
In collaboration with Professors Hinds and Kleinman, large scale collections of  particles and volatile components of ambient air were collected in Riverside, California using a Tisch Sampler with a 10 cm Teflon coated glass filter and a bed of XAD resin to collect PM2.5 and the corresponding vapor fraction, respectively.  The objective of the study was to conduct comprehensive analyses, from physical characterization to in vivo animal responses on these air samples.  By collecting both particle and volatile fractions, the chemical analyses would provide the chemical properties of the particle and vapor phase of an air sample. The collections represented a total of about 300 m3 of air, a sufficiently large volume to perform multiple assay procedures.  Preliminary studies of the two fractions showed that aqueous suspensions of the particulate fraction were more redox active than the volatile fraction and that electrophilic activity was higher in the vapor phase.   The inhibitory effects of the vapor fraction on protein tyrosine phosphatase1B (PTP1B) were also determined.  PTP1B, like GAPDH, is also a thiolate enzyme and can be inactivated by electrophiles in a similar manner.
 
Results:  This study of ambient air is the first, to our knowledge, in which chemical properties of the particle and vapor phase of the same air mass are being investigated.  The redox properties of the air mass in the three samples were predominately in the particle phase whereas the electrophiles were predominately in the vapor phase.  The observations made earlier with the DEPs would suggest that the redox activity in particle phase was particle bound.  The presence of electrophiles in the vapor phase indicate that they are of lower molecular weight and as quinone data collected in Riverside earlier showed that the naphthoquinones were present in high levels, they may be major consituents.  Since the TOF/MS results from the protein bound species indicate a molecular weight of 422 for the bound species, it is likely that multiple equivalents bind to PTP.  
 
Cellular studies showed that the electrophiles in air volumes of 1-2 m3 can activate the MAPkinase cascade which results in cell proliferation and cytokine expression.  In other cells, protective events can occur through the activation of the Nrf2-keap1 system.  Nrf2 is a transcription factor that when activated will initiate the synthesis of multiple protective proteins such as glutathione transferases, multidrug resistant proteins, quinone reductases and glutathione synthases.   Thus, components of the vapor phase can elicit both adverse and protective effects, depending on their content and concentrations.
 
The assays developed in this project provide quantitative estimates of both redox and electrophilic activity in a given sample.  In this period, their quantitative nature has been used to characterize the physical and chemical properties of the components responsible for the activities.  While the results do not provide information on the specific chemical species responsible for the effect, they can provide information on the chemical class involved. 
 
Results from studies with multiple DEP samples indicate that the redox activity, both metal- and organic-based, is particle bound in aqueous media.  In contrast, electrophilic species are water soluble and are present in significant levels in the vapor phase.  Support for the presence of quinone like compounds in the organic components was provided by reductive acetylation which converts quinones to their inactive hydroquinone esters.  This reaction, which inactivates quinones, caused a loss of both redox and electrophilic activity of the organic fraction of DEPs. The distribution of activities between particles and water are relevant to particle exposure, for they suggest that redox based reactions associated with particles will remain with the particles upon their deposition into lung tissue, whereas electrophiles will be more soluble.  Particle access to cells is variable, as multiple pathways are possible, but if the particle deposits on the cell surface, the redox reactions observed can take place on the cell surface, using electrons available from compounds such as ascorbate and glutathione, which are present in lung lining fluid.  In contrast, electrophiles appear to dissociate from the particles and can diffuse into cells.  Thus, the bioavailability of the redox active species in DEPs will be very different from that of electrophiles. Electrophiles also have distinctive exposure properties because they form covalent bonds with tissue nucleophiles such as protein thiols groups to inactivate them.  Depending on the turnover of the protein involved, the actions of these electrophiles can be cumulative and become significant with chronic exposure.
 
The initial studies of vapor phase samples from Riverside show that this phase contains substantial levels of electrophiles and emphasize the importance of this component of air pollution.  Experiments examining the cellular effects of the extracts showed that both inflammatory and protective responses occur, mediated by different cellular signaling pathways.  In summary, in the course of the first three years of project 3, we have developed and applied three assays for toxicologically relevant chemical reactivity in ambient air samples.  Their quantitative nature has allowed a comparison of activities of samples, reflecting differences in sites of collection, multiple collections at the same site, and fractionation of DEPs.
Re

Expected Results:

This Project will characterize ambient PM samples from key sources according to their reactivity in redox and electrophilicity assays, and in association with toxicological findings, will provide a basis for identifying PM of the greatest concern for public health in terms of potential to induce oxidative stress and related health effects.

Future Activities:

1.      The DEP studies showed that redox active metals are tightly bound to particles in an aqueous suspension.  One explanation was the notion that organic functions on the particles such as carboxyl groups may chelate metals.  To investigate this possibility, approaches to particle modification such as esterification will be examined.  In addition, DEPs doped with specific metals may be available from the EPA.  These particles would be particularly useful in planned studies of the availability of bound metals for participation in chemical reactions.
2.      The studies of particle and vapor phase components of ambient air will continue, initially with completion of studies of the particle phase of the current samples.  Then, collections will be made at different sites and at different seasons to determine the roles of these parameters in the reactivities of the two phases.

References:

1.      Cho, A., Di Stefano, E., Ying, Y., Rodriguez, C.E., Schmitz, D.A., Miguel, A.H., Eiguren-Fernandez, A., Kobayashi, T.E., Avol, E., and Froines, J.R. Determination of Four Quinones in Diesel Exhaust Particles, SRM 1649a and Atmospheric PM2.5. Aerosol Science and Technology 38:68-81, 2004.
2.      Claiborne, A., Yeh, J.I., Mallett, T.C., Luba, J., Crane, E.J., Charrier, V., Parsonage, D. Protein-sulfenic acids: diverse roles for an unlikely player in enzyme catalysis and redox regulation. Biochemistry 38:15407-15416, 1999.
3.      Ghio, A.J., Stonehuerner, J., Pritchard, R.J., Piantadosi, C.A., Quigley, D.R., Dreher, K.L., and Costa, D.L. Humic-like substances in air pollution particulates correlate with concentrations of transition metals and oxidant generation. Inhalation Toxicology 8:479-494, 1996.
4.      Poole, L.B., Karplus, P.A. and Claiborne, A. Protein sulfenic acids in redox signaling. Annual Reviews Pharmacology and Toxicology. 44:325-347, 2004.
5.      Steelink, C. What is humic acid.  Journal of Chemical Education 40:379-384, 1963.


Journal Articles on this Report : 8 Displayed | Download in RIS Format

Other subproject views: All 47 publications 27 publications in selected types All 27 journal articles
Other center views: All 241 publications 157 publications in selected types All 157 journal articles
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Journal Article Ayres JG, Borm P, Cassee FR, Castranova V, Donaldson K, Ghio A, Harrison RM, Hider R, Kelly F, Kooter IM, Marano F, Maynard RL, Mudway I, Nel A, Sioutas C, Smith S, Baeza-Squiban A, Cho A, Duggan S, Froines J. Evaluating the toxicity of airborne particulate matter and nanoparticles by measuring oxidative stress potential—a workshop report and consensus statement. Inhalation Toxicology 2008;20(1):75-99. R832413 (2008)
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  • Journal Article Eiguren-Fernandez A, Avol EL, Thurairatnam S, Hakami M, Froines JR, Miguel AH. Seasonal influence on vapor-and particle-phase polycyclic aromatic hydrocarbon concentrations in school communities located in Southern California. Aerosol Science & Technology 2007;41(4):438-446. R832413 (2008)
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  • Journal Article Eiguren-Fernandez A, Miguel AH, Lu R, Purvis K, Grant B, Mayo P, Di Stefano E, Cho AK, Froines J. Atmospheric formation of 9,10-phenanthraquinone in the Los Angeles air basin. Atmospheric Environment 2008;42(10):2312-2319. R832413 (2007)
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  • Journal Article Inoue K-I, Takano H, Ichinose T, Tomura S, Yanagisawa R, Sakurai M, Sumi D, Cho AK, Hiyoshi K, Kumagai Y. Effects of naphthoquinone on airway responsiveness in the presence or absence of antigen in mice. Archives of Toxicology 2007;81(8):575-581. R832413 (2007)
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  • Journal Article Iwamoto N, Sumi D, Ishii T, Uchida K, Cho AK, Froines JR, Kumagai Y. Chemical knockdown of protein-tyrosine phosphatase 1B by 1,2-naphthoquinone through covalent modification causes persistent transactivation of epidermal growth factor receptor. Journal of Biological Chemistry 2007;282(46):33396-33404. R832413 (2008)
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  • Journal Article Ntziachristos L, Froines JR, Cho AK, Sioutas C. Relationship between redox activity and chemical speciation of size-fractionated particulate matter. Particle and Fibre Toxicology 2007;4:5 (12 pp.). R832413 (2008)
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  • Journal Article Shinyashiki M, Rodriguez CE, Di Stefano EW, Sioutas C, Delfino RJ, Kumagai Y, Froines JR, Cho AK. On the interaction between glyceraldehyde-3-phosphate dehydrogenase and airborne particles:evidence for electrophilic species. Atmospheric Environment 2008;42(3):517-529. R832413 (2008)
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  • Journal Article Taguchi K, Shimada M, Fujii S, Sumi D, Pan X, Yamano S, Nishiyama T, Hiratsuka A, Yamamoto M, Cho AK, Froines JR, Kumagai Y. Redox cycling of 9,10-phenanthraquinone to cause oxidative stress is terminated through its monoglucuronide conjugation in human pulmonary epithelial A549 cells. Free Radical Biology and Medicine 2008;44(8):1645-1655. R832413 (2007)
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  • Supplemental Keywords:

    Bioavailability, metabolism, chemicals, oxidation-reduction, biochemistry, ambient air, particulate matter, oxidative stress,, RFA, Health, Scientific Discipline, Air, particulate matter, Health Risk Assessment, Risk Assessments, Biochemistry, Ecology and Ecosystems, particulates, atmospheric particulate matter, chemical assys, particle matrix, chemical characteristics, human health effects, PM 2.5, toxicology, airway disease, cardiovascular vulnerability, airborne particulate matter, air pollution, human exposure, vascular dysfunction, cardiovascular disease, human health risk

    Progress and Final Reports:

    Original Abstract
  • 2006 Progress Report
  • 2007 Progress Report
  • 2009 Progress Report
  • 2010 Progress Report
  • 2011
  • Final Report

  • Main Center Abstract and Reports:

    R832413    Southern California Particle Center

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R832413C001 Contribution of Primary and Secondary PM Sources to Exposure & Evaluation of Their Relative Toxicity
    R832413C002 Project 2: The Role of Oxidative Stress in PM-induced Adverse Health Effects
    R832413C003 The Chemical Properties of PM and their Toxicological Implications
    R832413C004 Oxidative Stress Responses to PM Exposure in Elderly Individuals With Coronary Heart Disease
    R832413C005 Ultrafine Particles on and Near Freeways