Grantee Research Project Results

2007 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, 2006 through September 30, 2007
RFA: Particulate Matter Research Centers (2004) RFA Text | Recipients Lists
Research Category: Human Health , Air

Objective:

To characterize the catalytic redox and electrophilic properties of ambient PM samples using cell-free chemical assays. The hypothesis being tested is that PM have constituents that are capable of inducing cellular stress by different chemical processes and that such processes can be quantitatively assessed by specific analytical chemical procedures.
To determine how chemical properties, cellular disposition and cellular actions of organic and inorganic compounds are affected by the particle matrix. The hypothesis being tested is that the particle matrix alters the physical chemical properties of the associated chemical species such that cellular availability is altered.

Progress Summary:

Chemical assays: This project has developed and continues to develop assay procedures that relate to the hypothesis and has applied them to samples from ambient air, diesel exhaust and synthetic particles. The chemical assays provide quantitative data on:

Electron transfer capacity of organic constituents, using the consumption of dithiothreitol (DTT) as the measure of activity.
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. This assay is still being developed; control studies evaluating the actions of low concentrations of metals are being performed.
The content of electrophilic species, capable of forming covalent bonds with a thiolate enzyme, GAPDH, in PM samples. The ability of the sample to inactivate the enzyme under anaerobic conditions is determined.
The change in oxidative status of cells during exposure. The induction of the stress protein hemeoxygenase-1 and the changes in the redox status of glutathione are measured.
Specific chemical species relevant to the operating hypothesis are also measured, specifically:

Polynuclear aromatic hydrocarbons
Selected Quinones
Metal ion species have been measured by inductively coupled plasma mass spectrometry.

Current results/accomplishments: New findings since 6/30/06

Evaluation of the DHBA procedure for redox active metal species.
The formation of DHBA is based on the generation of hydroxyl from hydrogen peroxide by the action of transition metal ions such as copper and iron. The hydrogen peroxide is generated by the action of ascorbic acid on redox active species, and is analogous to the interactions the may take place between PM and lung fluid, which has significant ascorbate levels. DHBA formation under the conditions of the assay is inhibited by the metal chelator, diethylenetriamine pentaacetic acid (DTPA). Redox active organic compounds such as quinones, which are active in the DTT assay, do not catalyze DHBA formation.

In this period, 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 showed that DHBA formation, i.e., Fenton reaction catalysis was linearly correlated with copper concentration but not with iron concentration. Redox active organic compounds such as quinones do not participate in the reaction. The DHBA formation rate for cupric sulfate alone, however, was found to be much lower (about 1/50^th) than that found in the samples, i.e., the copper concentration alone was insufficient to account for the observed rate of DHBA formation in the ultrafine particle suspensions. This discrepancy could be due to several factors, which are currently under investigation:

There are additional transition metals in the sample. However, levels of vanadium and manganese found were less than 1/10^th of copper and iron.
Organic material, as well as transition metals, catalyze the reduction of oxygen to hydrogen peroxide in the assay system. Since the rate of the Fenton reaction is proportional to hydrogen peroxide concentration, generation of hydrogen peroxide from redox active organic compounds could result in increased DHBA formation.
There could be more complex interactions between the particles and metals that increase the catalytic capacity of Cu in the Fenton reaction.

The presence of electrophiles in PM samples
An alternative mechanism for oxidative stress induction is by covalent bond formation between electrophilic PM components and the cellular nucleophiles, most notably thiols. Reactions between electrophiles and protein thiols can irreversibly inactivate them. Proteins involved in critical cellular functions such as transport, signal transduction and metabolism utilize the redox properties of their thiols in their actions, which would be blocked by such covalent modification. These covalent changes are irreversible and can be cumulative during chronic exposure, even to very low levels of electrophiles, and lead to pathological changes in the affected tissues over time. We have used the thiol protein, GAPDH, as a target nucleophile for electrophilic species in a given test sample and allowed the enzyme to be inactivated under anaerobic conditions to avoid oxygen based inactivation. We have measured 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 reductases (Claiborne et al., 1999; Poole et al., 2004).

Cellular uptake of PM and their constituents
Studies of cellular uptake of PM constituents used the ability of cells to metabolize organic compounds. Cells take up organic compounds by transport and simple diffusion, with the contributions of each highly dependent on their chemical structure and physico-chemical properties. Once entering the cell, the compound is metabolized by numerous enzymes. By monitoring metabolites of representative compounds in the presence and absence of particles, the effect of particles uptake can be assessed quantitatively.

In our initial studies, quinones were used as surrogates of reactive organic compounds of PM. We have found that the rate limiting step in 9,10-phenanthroquinone (PQ) metabolism is due to a dicoumarol-sensitive quinone reductase. We are currently developing assays for the metabolism of 1,4-benzoquinone and 1,2- and 1,4-naphthoquinones to monitor the rate limiting steps of their metabolism.

Future Activities:

The validation of the analytical procedures and their utility will depend on the ease of reproducibility by other investigators and understanding the nature of the chemical species that are detected. We have entered into collaborative studies with the EPA to study particles of different sources and properties to determine the relationship between the chemical assays and toxicological measurements. We have received particles from such as diesel exhaust and artificial carbon particles from the laboratory of Dr. William Linak and are currently evaluating their performance in our analytical procedures.
Analysis of particle samples collected in Project one will continue.
Studies of cellular uptake, monitored by metabolism, will utilize macrophages and epithelial cells.
Special collections, such as those described in Project 5 will be analyzed to determine time dependent changes in activity.
Large scale collections are planned for cellular toxicity and chemical characterization of volatile constituents of ambient air.

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

Publications Views
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

Publications
Type	Citation	Sub Project	Document Sources
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) R832413 (Final) R832413C003 (2007) R832413C003 (2008) R832413C003 (Final) R827352 (Final) R827352C009 (Final) R827352C013 (Final)	Full-text: Taylor&Francis-Full Text HTML Exit Abstract: Taylor&Francis-Abstract Exit Other: Taylor&Francis-Full Text PDF Exit
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) R832413 (2008) R832413 (Final) R832413C003 (2007) R832413C003 (2008) R832413C003 (2009) R832413C003 (2010) R832413C003 (Final) R827352 (Final)	Full-text: ScienceDirect-Full Text HTML Exit Abstract: ScienceDirect-Abstract Exit Other: ScienceDirect-Full Text PDF Exit
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) R832413 (2008) R832413 (Final) R832413C003 (2007) R832413C003 (2008) R832413C003 (2010)	Abstract from PubMed Abstract: Springer-Abstract Exit
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) R832413 (Final) R832413C003 (2007) R832413C003 (2008) R832413C003 (2010) R832413C003 (Final)	Abstract from PubMed Full-text: JBC-Full Text HTML Exit Abstract: JBC-Abstract Exit Other: JBC-Full Text PDF Exit
Journal Article	Kleinman MT, Sioutas C, Froines JR, Fanning E, Hamade A, Mendez L, Meacher D, Oldham M. Inhalation of concentrated ambient particulate matter near a heavily trafficked road stimulates antigen-induced airway responses in mice. Inhalation Toxicology 2007;19(Suppl 1):117-126.	R832413 (2008) R832413 (2009) R832413 (Final) R832413C001 (2007) R832413C001 (2008) R832413C001 (Final) R832413C003 (2007) R827352 (Final)	Abstract from PubMed Abstract: Taylor and Francis-Abstract Exit
Journal Article	Krudysz MA, Froines JR, Fine PM, Sioutas C. Intra-community spatial variation of size-fractionated PM mass, OC, EC, and trace elements in the Long Beach, CA area. Atmospheric Environment 2008;42(21):5374-5389.	R832413 (2007) R832413 (2008) R832413 (Final) R832413C001 (2007) R832413C001 (Final) R832413C003 (2007) R832157 (2007) R832157 (Final)	Full-text: ScienceDirect-Full Text HTML Exit Abstract: ScienceDirect-Abstract Exit Other: ScienceDirect-Full Text PDF Exit
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) R832413 (2009) R832413 (Final) R832413C001 (2008) R832413C001 (Final) R832413C003 (2007) R832413C003 (2008) R832413C003 (2010) R832413C003 (Final) R832413C004 (2009) R832413C004 (2010)	Full-text: ScienceDirect-Full Text HTML Exit Abstract: ScienceDirect-Abstract Exit Other: ScienceDirect-Full Text PDF Exit
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) R832413 (2008) R832413C003 (2007) R832413C003 (2008) R832413C003 (2009) R832413C003 (2010) R827352 (Final)	Abstract from PubMed Full-text: ScienceDirect-Full Text HTML Exit Abstract: ScienceDirect-Abstract Exit Other: ScienceDirect-Full Text PDF Exit

Supplemental Keywords:

RFA, Health, Scientific Discipline, Air, particulate matter, Health Risk Assessment, Risk Assessments, Biochemistry, Ecology and Ecosystems, atmospheric particulate matter, particulates, chemical assys, particle matrix, human health effects, PM 2.5, chemical characteristics, toxicology, airway disease, airborne particulate matter, cardiovascular vulnerability, air pollution, human exposure, vascular dysfunction, cardiovascular disease, human health risk

Progress and Final Reports:

Original Abstract

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

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.