Final 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. , Harkema, Jack , Fukuto, Jon , Kumagai, Yoshito
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)
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Health Effects , Air

Objective:

Background

The Committee on Research Priorities for Airborne Particulate Matter of the National Academy of Sciences has pointed out that while studies of the chemical components of PM have focused on transition metals, there is increasing evidence that organic compounds, bound or condensed onto PM, also contribute to the adverse health effects. They also point out that more information is needed on the roles and mechanisms of these organic compounds in PM or co-pollutants (Topic 5, (Samet and Others, 2004)). Earlier observations had proposed oxidative stress as the key event leading to the adverse health of air pollution (Baeza-Squiban et al., 1999; Frampton et al., 1999; Li et al., 2000; Nel et al., 2001). The state of oxidative stress refers to a cellular condition characterized by a change in the relative concentrations of the oxidized form of redox active agents such as glutathione (GSH) and nicotinamide nucleotides (NADPH and NADH) (Schafer and Buettner, 2001). This state can be caused by increasing intracellular concentrations of reduced oxygen species such as superoxide, hydrogen peroxide and hydroxyl radical.  These so called reactive oxygen species (ROS) are typically generated by electron transfer reactions between oxygen and an electron source which are catalyzed by prooxidants such as transition metals and quinones. It should be pointed out, however, that the ratio of oxidized glutathione (GSSG) to GSH can also be altered by loss of GSH to electrophilic reactions in which GSH is covalently bound to electrophile such as quinones and α,β unsaturated carbonyl compounds (see for example, (Rodriguez  et al., 2004)). 

In project 3 of the Southern California Particle Center, we have studied the chemical components and chemical properties of particulate and volatile samples of ambient air in an attempt to relate these parameters to observed biological effects. Thus, we have measured the quantities of polynuclear aromatic hydrocarbons (PAHs), precursors to highly reactive chemical species that can participate in cellular reactions that lead to oxidative stress. We found levels of two and three ring aromatic hydrocarbons to be quite high, with naphthalene and phenanthrene at levels 100 to 10,000 times those of other PAHs (Eiguren-Fernandez et al., 2004). These lower molecular weight PAHs are particularly important because they can be converted to the toxic quinones by combustion, bioactivation (Sheets et al., 2004)and by atmospheric chemical processes (Barbas et al., 1996; Sasaki et al., 1997). Another recent study demonstrated similar high levels of the naphthalenes in volatile fractions of diesel exhaust and concluded that the immunotoxicity associated with diesel exhaust was not due to the higher molecular weight PAHs such as benzo[a]pyrene and its quinones but due to the lower molecular weight compounds such as naphthalene, methyl naphthalene and their oxygenated derivatives (Burchiel et al., 2004). Professor Gilliland and his coworkers of the USC Children’s Health Study Project have reported a finding that we believe to be particularly relevant to the current project (see (Salam et al., 2006.)). These workers have found that the incidence of asthma in children living in the Los Angeles Basin is greater among those with genetically higher levels of epoxide hydrolase, a key enzyme in the conversion of aromatic hydrocarbons such as naphthalene to the corresponding quinones. Thus, although levels of lower molecular weight PAHs are high in diesel exhaust and in the Los Angeles Basin, the immediate adverse effects, such as exacerbation of asthma, is likely due to their oxidation products which are formed in the combustion process, in the atmosphere or by biotransformation reactions in the host organism.

Operating hypotheses

Our hypothesis is that acute adverse effects of air pollutants could be accounted for by chemical species capable of either or both of two basic reactions, prooxidant and electrophilic. Numerous studies have demonstrated the inflammatory actions of ambient air and diesel exhaust particles (Baeza-Squiban et al., 1999; Veronesi et al., 1999; Samet et al., 2002). These actions are associated with the exacerbation of pulmonary (Pandya et al., 2002; Riedl and Nel, 2008), cardiovascular (Donaldson et al., 2001; Araujo et al., 2008)} and neurological disease (Block and Calderon-Garciduenas, 2009; Gerlofs-Nijland et al., 2010; Genc et al., 2012)states. However, the cellular responses to such particles and air samples are both pro and anti-inflammatory, i.e., when cells are exposed to particles and vapors, transcription factors associated with inflammation (Ng et al., 1998; Takizawa et al., 1999)and with antiinflammation, or adaptation (Baulig et al., 2003; Li et al., 2004), are activated simultaneously. The overall effect of ambient air will be determined by the nature and quantities of the responsible chemical species. Ambient air particulates are a complex mixture of metals and reactive organic compounds. Transition metals are prooxidants, i.e., they generate reactive oxygen species such as superoxide and hydrogen peroxide through reactions with cellular antioxidants such as ascorbate. Quinones are prooxidants but are also electrophilic, i.e., they can form covalent bonds with protein side chain groups such as thiols and imidazoles (Monks and Lau, 1992; Troester et al., 2002; Iwamoto et al., 2007). Other electrophiles such as organic compounds with α,β-unsaturated carbonyl functions are also present in ambient air samples (Jakober et al., 2006). These chemical reactions, the formation of reactive oxygen by prooxidants and the formation of covalent bonds by electrophiles, are the major reactions associated with the adverse health effects of ambient particles, for as prooxidants they can generate reactive oxygen species (ROS) that are associated with oxidative stress and, as electrophiles, can react with nucleophilic thiols to form covalent bonds with key biological molecules.  

Prooxidant air pollutants will generate reactive oxygen species (ROS) such as superoxide, hydrogen peroxide and hydroxyl radical, using cellular reducing agents or antioxidants. These ROS can convert protein thiolates to their sulfenates, and initiate cellular cascades that lead to inflammatory and adaptive responses.  Electrophilic pollutants can conjugate with thiol proteins in an irreversible reaction, thereby inactivating the protein. This reaction sequence activates the inflammatory and adaptive pathways but with different time constants. The ROS based effects are reversible; the cell can reverse thiol oxidation with antioxidants such as NADPH and glutathtione (GSH).  In contrast, electrophile based inactivation is irreversible and recovery from this insult requires the cell to resynthesize the affected protein. As a result, chronic exposure to low doses of electrophiles can be cumulative, especially when the affected protein has a long half life. For example, the half life of the common protein, albumin, is of the order of 20 days, so it will require ~60 days to completely turnover the albumin pool, so during chronic exposure electrophile conjugation can gradually increase to a steady state.

One of the key targets of these reactive chemical species are cellular thiols. Transcription factor regulators and many other regulator proteins contain cysteine thiols that are readily oxidized to the sulfenic acid state by ROS, most notably hydrogen peroxide. This process is reversed in cells by a chemical reaction involving glutathione as the reducing agent and is the major pathway for activation and inactivation of these proteins. Thiols are also nucleophilic and will form covalent bonds with electrophilic compounds such as quinones and α,β unsaturated carbonyl compounds. In this case, recovery of the thiols requires resynthesis of the irreversibly modified protein, which could have a lengthy time constant.    

Summary/Accomplishments (Outputs/Outcomes):

Determination of reactive chemical species content of air pollutant mixtures

To test our hypothesis, we attempted to demonstrate ;

1. The presence of reactive organic chemical species in air pollutant mixtures.

2. The ability of air pollutant mixtures to participate in toxicologically relevant reactions.

3. The ability of cells, when exposed to air pollutants, respond in a dose dependent manner with changes consistent with the reported health effects. 

We first developed a quantitative assay for 4 quinones likely to be present in air pollutant (Cho et al., 2004).  Quinones are a class of organic compounds that exhibit prooxidant and electrophilic properties and therefore represent potential candidates for the reactive species present in air pollution.  

Next, to determine whether prooxidants were present in air pollutant mixtures, we developed an assay that quantitates the ability of a test sample to generate reactive oxygen from dithiothreitol (DTT), i.e., transfer electrons from DTT to oxygen to generate the reduced and reactive species. The assay has been used to relate activity to cellular stress induction (Li et al., 2003b) and to in vivo responses (Li et al., 2003a; Cho et al., 2005). After its initial publication, this assay has been extensively used by other investigators for evaluation of potential toxicity and for characterization of reactive mixtures in air pollutant samples (Chung et al., 2006; Hu et al., 2008; Biswas et al., 2009; Verma et al., 2009; Lin and Yu, 2011; Charrier and Anastasio, 2012; Vishal Verma et al., 2012) thereby providing an increasing data base of prooxidants from a variety of sources and a comparison with other properties relevant to air pollution toxicity.  

We also developed an electrophile assay, based on the ability of the sample components to irreversibly inhibit the thiol enzyme, glyceraldehydes-3-phosphate dehydrogenase (GAPDH) to provide a quantitative measure of electrophile content. The thiol group on a key cysteine moiety in GAPDH forms a covalent bond with electrophiles, in contrast to peroxides whose action is reversed by the reactive thiolate, DTT (Shinyashiki et al., 2008). Although the enzyme appears to have a wide range of substrate/inhibitor structures, they have not been strictly assessed so the assay is limited by this characteristic. 

Studies of diesel exhaust particles

Using these assay procedures we have characterized diesel exhaust particles from Japan (Cho et al., 2004; Cho et al., 2005) and the EPA (Shinyashiki et al., 2008; Shinyashiki et al., 2009a) and will examine diesel exhaust from UC Riverside, collected under carefully controlled conditions. We have used and continue to use the DEP from Japan (J-1 DEP) as a standard in all of the assays we have developed. First, quinone content was determined in the samples as shown below:

Sample

1,2-Naphthoquinone

1,4- Naphthoquinone

9,10-Phenanthroquinone

Ref

J-1 (Japan)

22.3

19.9

18.7

(Cho et al., 2004)

DEP-4 (EPA)

26.0

23.0

14.0

(Shinyashiki et al., 2009a)

DEP-3 (EPA)

1.77

1.26

8.05

(Shinyashiki et al., 2009a)

Concentrations in µg/mg sample. 

Then, the chemical reactivity was assessed with the DTT and GAPDH assays:

Sample

DTT pmoles DTT consumed/micg

% inhibited by DTPA*

GAPDH activity/micg

Ref

J-1 (Japan)

34

 

32.2

 

DEP-4 (EPA)

15

41%

28.7

(Shinyashiki et al., 2009a)

DEP-3 (EPA)

7.8

65%

7.86

(Shinyashiki et al., 2009a)

*DTPA is a metal chelator; the % inhibited is the % of the reaction presumably catalyzed by metals

The tables summarize results of the multiple assays performed on the samples. There was considerable variation in the EPA samples; two samples representing the extreme values for concentration of quinones are shown.  

Their presence demonstrates that quinones are products of the combustion process as well as atmospheric processes (Eiguren-Fernandez et al., 2008c). In a study of the distribution of the distribution of the 4 quinones between the particle and vapor phase in ambient air samples in the Los Angeles Basin, the naphthoquinones were found to be concentrated in the vapor phase while 9,10 phenanthroquinone was mostly present in the particle phase (Eiguren-Fernandez et al., 2008a). DEP-4 had a high OC/EC ratio (0.6) compared to DEP-3 (0.3), suggesting that the prooxidant and electrophile content was mostly organic in nature. The inhibition by DTPA also indicated that DEP-4 had higher levels of metal based prooxidants than DEP-3. The GAPDH assay for electrophiles was also reflective of the organic content and the similar quione content of DEP-4 and J-DEP suggests that compounds such as the naphthoquinones are the electrophiles. In related studies Daher et al., (Daher et al., 2011) have shown that placement of a thermal denuder that traps organic compounds before exhaust stream passes through the filters during DEP collection eliminates essentially all of the DTT active materials, indicating that the reactive species in diesel exhaust are volatile organics. This observation is highly relevant to studies of ambient air.    

Further information on the nature of the reactive organic species in DEPs was provided by chemical studies of an organic extract of DEP-4 (Shinyashiki et al., 2009b). Dichloromethane extracts of this material were subjected to the zinc dust/acetic anhydride reduction process used to convert quinones to their more stable acetylated hydroxy derivatives (Cho et al., 2004). This procedure resulted in a preparation devoid of prooxidant activity and with only 16% of the electrophilic activity. Of organic functional groups most likely to be affected by this procedure are quinones or quinoid structures, but α,β-unsaturated carbonyls may also be converted to their unreactive carbinols, but only quinones would exhibit prooxidant activity. 

Although DEP may be considered an artificial preparation compared to ambient aerosols, their studies are vital to our understanding of air pollution toxicity because they are available in quantities sufficient for use in the development of quantitative assay procedures to be used in ambient air investigation and in vivo studies. The data obtained in project 3 suggest that the quinoid type organic components of DEP are associated with the electrophile content since differences in the quinone content of the EPA DEP are reflected more by the GAPDH activity. DEP are extremently complex however, as analysis for specific chemicals have shown.  In an early study investigating the contribution of quinones to overall prooxidant content in ambient air samples, we were surprised to note that at best, only about 20% could be accounted for by the quinones assayed (A.K. Cho unpublished results). A possible explanation for this descrepancy may be the presence of humic like substances in diesel exhaust and in ambient particles (Ghio and Quigley, 1994; Lin and Yu, 2011).  Humic like substances are polymers or oligimers of hydroxylated aryl- and aryl-alkyl acids (Steelink, 1963)and are found mostly in the untreated water of rivers and ponds (Collins et al., 1986). They are thought to be generated by the decomposition of lignins but their presence in air pollutant mixtures, demonstrated by their solubility in aqueous alkaline mixtures and separation by acidification, suggests they can be formed by the high temperatures and conditions of fuel combustion and condense onto the particle core upon cooling in the atmosphere. These substances contain α,β-unsaturated carbonyls, catechols, dicarboxylic acids and other polar functions capable of prooxidant, electrophilic and metal chelating activity.  Recently, Lin et al., (Lin and Yu, 2011) have shown that they can be extracted from ambient parties with aqueous solvents and that they exhibit DTT assay based prooxidant activity. We are currently examining  humic acids obtained commercially with our chemical and cellular assays to evaluate these substances as potential reactive species in ambient aerosols. 

Cellular studies of DEP

We are establishing quantitative assay procedures for cellular markers characteristic of the inflammatory and adaptive/protective response using the response to J-DEP exposure by a murine macrophage cell line, Raw 264.7 as a starting point. This cell line has been extensively used by air pollution investigators to evaluate the toxicity of air pollutants  as it relates to the exacerbaton of cardiovascular and pumonary diseases. These cells respond to reactive chemical species by intiating both inflammatory and adaptive, or protective responses. These responses are mediated by the transcription factors, NFκB and or Nrf2, respectively, which, in turn, induce the expression of tumor necrosis factor alpha (TNFα) and hemeoxygenase (HO-1). The latter two proteins can be quantitatively assayed with enzyme-linked immunosorbant assays (ELISA). In our preliminatry studies with J- DEP, we have found that under conditions of minimal toxicity (10 µg/mL) the primary response from a dichloromethane extract of these DEP appears to be one of adaptation or protection, evidenced by the greater increase in HO-1 compared to TNFα. Thus, incubation of the extract of J-DEP with the cells caused increases in TNFα and HO-1 by 3 and 352 ng/mL of media, respectivly. For comparison, preliminary data from studies of ambient particles and vapors (see below) have shown that particles themselves (not extracts) increase TNFα by 48 ng/mL and vapors corresponding to the particles increase HO-1 by 27.6 ng/mLat concentrations of 1 m3/mL. At this concentration, the particles exhibit no significant toxicity beyond that caused by the water extract of the teflon coated filters from which the particles were obtained. 

We will extend our findings of DEP with samples obtained under controlled conditions by colleagues at The Center for Environmental Research and Technology at The University of California, Riverside. These DEP and their corresponding vapors will be fractionated according to their physical properties by using solvents of varying polarity and the fractions assayed for chemical classes (PAH, quinones, carbonyls, metals) as well as prooxidants and electrophiles. Cellular assays will be performed on the fractions was well to characterize properties of proinflammatory and adaptive agents in DEP.

Properties of particles and their corresponding vapor phase in ambient air. 

In addition to collaborative studies with investigators in Project 2 (IS THIS COSTAS’ SECTION?) We have examined particle (PM2.5) and vapor phases from several other sites in the Los Angeles basin for prooxidant and electrophile content. These studies used large scale collections made continuously over a period of several days, using a Tish Sampler, a high volume device with a filter and XAD resin trap below to absorb volatile organic species (Eiguren-Fernandez and Miguel, 2003; Eiguren-Fernandez et al., 2004; Eiguren-Fernandez et al., 2007; Eiguren-Fernandez et al., 2008a). The limitations of this mode of collection include:

  1. The loss of day-night differences in air pollution content due to changing wind patterns, temperature and sunlight. 
  2. Loss of particle bound volatile species to the vapor phase as the air continuously passes through the filters. However, most of the volatile organic matter is presumed to be absorbed by the XAD resin, a polystyrene based material into which vapor phase organics would be absorbed.
  3. Changes in the composition of the particle phase as a result of the continuous flow of oxygen over the particles.  
  4. As collected the vapor phase is organic in nature, as the volatile inorganic gases are not collected. 

In exchange for these limitations however, there are some advantages:

  1. Most important is the ability to collect a large sample of ambient aerosol suitable for the multitude of assays to be performed.  Although not sufficient for extensive animal studies, the sample size is sufficient for our chemical reactivity and cellular studies. 
  2. The sample is time averaged over the collection time.  Even the use of these long collection times provided quite variable values for the same site a week later and limit the effective statistical analyses. 
  3. The collection provides two separate phases which can be independently examined.  Examination of the literature revealed very few studies examining the vapor phase although it is clearly important to understanding exposure to ambient aerosols. 

We have collected ambient aerosols in three separate studies, in Riverside, CA (Eiguren-Fernandez et al., 2010), in three communities adjacent to rail yards (in preparation) and in multiple sites neighboring the Commerce rail yards. The collections utilized Tisch medium-vol samplers with filters above XAD resin beds. The filters trapped PM2.5 and the XAD resin beds were used to trap volatile organic species which were extracted from the resins with dichloromethane. In general, the collections exhibited similar qualitative properties, i.e., the particles contained most (>80%) of the prooxidants with varying proportions of metals and the vapor contained most of the electrophiles (>80%). Of the communities studied, the summer vapor samples from San Bernardino consistently exhibited the highest levels of electrophiles with Riverside next and Long Beach and Commerce following. Prooxidant content was higher in Riverside compared to San Bernardino, Long Beach and Commerce. One feature of ambient PM that differed from DEP was the higher proportion of metal based prooxidants.  Inclusion of the metal chelator, DTPA in the DTT assay resulted in substantial reductions in DTT activity for all sites, in some cases such as those for Commerce, completely. Thus, most of the particle phase of ambient aerosols appears to contain metals whereas DEP include higher proportions of organic species. DEP are mostly made up of fuel combustion products with some metal components whereas ambient particles may include metals from vehicle brakes as well as commercial activities such as smelters. This difference also affects the nature of the cellular response to the different particles because of toxicokinetic issues. 

Many organic compounds can enter cells by passive diffusion because of the solubility in the lipid mambranes that make up the barrier between physioloical compartments which limits the entry of higly polar compounds. Non polar organic compounds entering cells by passive diffusion can initiate reactions intracellularly. Thus, prooxidant compounds such as 9.10 phenanthroquinone can use electrons from sources such as NADPH and GSH to generate the reactive oxygen species and electrophiles can form covalent bonds with different cellular nucleophiles to intiate adverse or adaptive changes in the cell. This barrier limits the access of polar compounds such as polyhydroxylated organic compounds and charged inorganic species which are transported. Intracellular concentrations of metals such as copper(Kaplan and Lutsenko, 2009) and iron (Anderson and Vulpe, 2009) are, on the other hand, carefully regulated by processes involving changes in their oxidation state and formation of complexes with inter- and extracellular proteins. While metals such as these have limited access to intracellular space, their presence in extracellular space such as the lung lining fluid can have effects on cells with which they interact. Lung lining fluid contains multiple redox active chemicals, including significant levels of ascorbate (Sun et al., 2001; Mudway et al., 2004) which can serve as the electron source for the reduction of oxygen to reactive species such as superoxide, hydrogen peroxide and hydroxyl radical.  These ROS can attack membranes increasing their permeability and initiating further chemical insults. 

Our first cellular study examined the effects of vapor phase components on Raw 264.7 cells (Iwamoto et al., 2010). At concentrations equivalent to ~ 2 m3/mL, the vapor components activated the epidermal growth factor (EGFR) and one of its downstream proteins, ERK, a member of the MAP kinase family, and the transcription factor, Nrf2. The MAP kinases activate the transcription factor, NFκB, and increase the expression of inflammatory cytokines (Abdelmohsen et al., 2003; Pourazar et al., 2008). The transcription factor Nrf2 is responsible for many adaptive responses to chemical toxins, acting through the antioxidant/electrophile response element (ARE) to trigger expression of proteins such as HO-1, glutathione transferases and glutathione synthases (Dinkova-Kostova et al., 2002; Wakabayashi et al., 2004) which protect the cell against chemical insults. These responses occurred in the absence of changes in the oxidative state of the cell which was examined by dichlorofluorescin fluorescence. Thus, the study showed that the vapor phase components from Riverside aerosols activated both proinflammatory and adaptive pathways at concentrations equivalent to 2 m3/mL by processes that did not affect the oxidative state of the cell. In a subsequent study, one of the vapor phase samples was exposed to cells from a human bronchial epithelial cell line (BEAS-2B) and the cell extract subjected to microarray analysis. The results showed that mRNA associated with activation of the ARE were increased with little or no response from proinflammatory mRNAs. These effects were reduced when the cells were exposed in the presence of high levels of glutathione, indicating that the effects were primarily due to electrophiles in the samples (Shinkai et al., in preparation). 

In summary, the results of initial studies of the vapor phase of ambient aerosols from Riverside, CA showed effects caused by electrophiles which were primarily adaptive, i.e., with minimal proinflammatory actions.  

Cell studies with Raw 264.7 cells of the air samples from the three railyard sites and communities near the Commerce Rail yard included both particles and vapor samples. The assays included the MTT based toxicity assay and ELISA assays for inflammation (TNF α) and adaptation (HO-1). The MTT assay uses the reduction of (3-[4,5-dimethylthiazol-2yl-])-2,5-diphenyl tetrazolium (MTT), to a purple formazan dye by active mitochondria in a cell preparation to determine the content of viable cells.  At 1 m3/mL, the highest concentration used for exposure, neither the particle nor the vapor samples exhibited significant toxcity. Concentration dependant changes in the levels of TNFα were observed for the particle samples from all sites at concentrations between 0.1 and 1.0 m3/mL with no significant actions by the vapors on this protein.  In contrast, the vapors induced HO-1 in a concentration dependent manner over the same concentration range, with no significant effect on TNFα expression. 

Prooxidant levels in samples collected from railyards in Commerce, Long Beach and San Bernardino varied with the community, but were always higher in the winter than summer. When all data were pooled, the vapor electrophile content correlated with vapor prooxidant content. When the results of the first week’s data were compared with gathered biological data, the determining factor appeared to be the vapor phase electrophile content, which correlated with HO-1 induction. TNF α induction did not correlate with proxidant content. It should be pointed out, however, that the limited correlations reflect only 3 values for each variable.

The third study examined the ambient aerosols over communities surrounding the Commerce rail yard. Prooxidant content clearly reflected proximity to the rail yard, with the background value about 1/5th of those at sites adjacent to the rail yard. Similar differences were noted between the background and rail yard electrophiles. Cell studies resulted in similar patterns of responses, particles increasing TNFα and vapors increasing HO-1 with minimal overlap. The initial cell study used samples from 4 sites. The induction of TNFα correlated with particle GAPDH and HO-1 induction correlated with both vapor prooxidant and electrophile content but not particle prooxidant content. While these correlations are limited because of the number of samples (8), the common factor appears to be the organic component content of both phases as the major contributor to the observed responses.  

In summary, studies of the particle and vapor phase of ambient aerosols collected in the Los Angeles Basin have shown the following:

1. That prooxidants are found almost completely in the particle phase and in the particle phase mostly metal based, as shown by sensitivity to DTPA.

2. As the vapor phase is collected on XAD resins and extracted with dichloromethane or other organic solvemt, the contents are organic. These compounds exhibit high levels of electrophilic activity with some prooxidant activity.

3. Particles cause a concentration dependent increase in the expression of the proinflammatory cytokine, TNFα, with minimal or no effect on HO-1, while the vapors cause a concentration dependent induction of HO-1. 

Thus, the aerosols contain significant levels of reactive chemical species, capable of inducing both inflammatory and adaptive responses by cells under conditions that are not toxic to the cell. However, these responses appear to correlate more with the organic components of particles and the vapors. This result would further suggest that the particles contain organic species that differ from those in the vapors and which are proinflammatory, not adaptive. There are at least two possible explanations for this result,

1. The organic species associated with particles are prooxidant in character. One of the more active prooxidants, 9,10-phenanthroquinone is mostly found in the particle phase, while the more electrophilic naphthoquinones are found in the vapor phase (Eiguren-Fernandez et al., 2008b). 

2 Ambient particles may contain significant levels of humic like substances (HLS) (Ghio et al., 1996; Shinyashiki et al., 2009b) which are both redox and electrophilically active. These species would have different toxicokinetic properties and the actions on cells could be different because of the manner they act on or enter cells.

Conclusions:

1. We have developed new assays to characterize air pollution in terms of toxicologically relevant reaction capabilities. These assays have been used to study DEP and ambient aerosols.

2. Studies of DEP indicate that while both metal and organic prooxidants are present, the organic components appear to be more relevant to the biological effects. 

3. Studies of ambient aerosols show that particles are primarily prooxidative, inducing TNFα while vapors are primarily electrophilic and induce HO-1. 

4. The organic material associated with particles may include HLS which exhibit both prooxidative and electrophilic properties.

Future studies will further characterize the chemical nature of prooxidants and electrophiles together with their biological effects. We will also attempt to identify the molecular targets of air pollutants by further characterization of the cellular responses, examining transcription factors and gene expression.   

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Nel AE, Diaz-Sanchez D and Li N., 2001. The role of particulate pollutants in pulmonary inflammation and asthma: evidence for the involvement of organic chemicals and oxidative stress. Curr Opin Pulm Med 7, pp.20-26.
 
Ng D, Kokot N, Hiura T, Faris M, Saxon A and Nel A.,1998.  Macrophage activation by polycyclic aromatic hydrocarbons: evidence for the involvement of stress-activated protein kinases, activator protein-1, and antioxidant response elements. J Immunol 161, pp.942-951.
 
Pandya RJ, Solomon G, Kinner A and Balmes JR.,2002. Diesel exhaust and asthma: hypotheses and molecular mechanisms of action. Environ Health Perspect 110, pp.103-112.
 
Pourazar J, Blomberg A, Kelly FJ, Davies DE, Wilson SJ, Holgate ST and Sandstrom T.,2008. Diesel exhaust increases EGFR and phosphorylated C-terminal Tyr 1173 in the bronchial epithelium. Part Fibre Toxicol 5, pp.8.
 
Riedl MA and Nel AE.,2008. Importance of oxidative stress in the pathogenesis and treatment of asthma. Curr Opin Allergy Clin Immunol 8, pp.49-56.
 
Rodriguez  C, Shinyashiki M, Froines J, Yu R, Fukuto J and Cho A.,2004.  An Examination of Quinone Toxicity using the Yeast Saccharomyces cerevisiae Model System. Toxicology 201, pp.185-196.
 
Salam M, Lin P, Gauderman W and Gilliland F.,2006. Genetic polymorphisms in microsomal epoxide hydrolase and childhood asthma.  Proc Am Thoracic Soc 3, pp.A349.
 
Samet JM and Others.,2004. Research priorities for airborne particulate matter: IV Continuing research progress. National Academy Press, Washington, DC.
 
Samet JM, Silbajoris R, Huang T and Jaspers I.,2002. Transcription factor activation following exposure of an intact lung preparation to metallic particulate matter. Environ Health Perspect 110, pp.985-990.
 
Sasaki J, Aschmann SM, Kwok ESC, Atkinson R and Arey J.,1997. Products of the Gas-Phase OH and NO<sub>3</sub> Radical-Initiated Reactions of Naphthalene. Environ. Sci. Technol. 31, pp.3173-3179.
 
Schafer FQ and Buettner GR.,200.  Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 30, pp.1191-1212.
 
Sheets PL, Yost GS and Carlson GP.,2004.  Benzene metabolism in human lung cell lines BEAS-2B and A549 and cells overexpressing CYP2F1. J Biochem Mol Toxicol 18, pp.92-99.
 
Steelink C.,1963. What is humic acid?. Journal of Chemical Education 40, pp.379-384.
 
Sun G, Crissman K, Norwood J, Richards J, Slade R and Hatch GE., 2001. Oxidative interactions of synthetic lung epithelial lining fluid with metal-containing particulate matter. Am J Physiol Lung Cell Mol Physiol 281, pp.L807-815.
 
Takizawa H, Ohtoshi T, Kawasaki S, Kohyama T, Desaki M, Kasama T, Kobayashi K, Nakahara K, Yamamoto K, Matsushima K and Kudoh S.,1999. Diesel exhaust particles induce NF-kappa B activation in human bronchial epithelial cells in vitro: importance in cytokine transcription. J Immunol 162, pp.4705-4711.
 
Troester MA, Lindstrom AB, Waidyanatha S, Kupper LL and Rappaport SM.,2002. Stability of hemoglobin and albumin adducts of naphthalene oxide, 1,2-naphthoquinone, and 1,4-naphthoquinone. Toxicol Sci 68, pp.314-321.
 
Veronesi B, Oortgiesen M, Carter JD and Devlin RB.,1999.  Particulate matter initiates inflammatory cytokine release by activation of capsaicin and acid receptors in a human bronchial epithelial cell line. Toxicol Appl Pharmacol 154, pp.106-115.
 
Vishal V Their 2 Hydrophobic/Hydrophilic Subfractions to the Reactive Oxygen 3 Species-Generating Potential of Fine Ambient Aerosols. Environ. Sci Technol.
 
Wakabayashi N, Dinkova-Kostova AT, Holtzclaw WD, Kang MI, Kobayashi A, Yamamoto M, Kensler TW and Talalay P.,2004. Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers. Proc Natl Acad Sci U S A 101, pp.2040-2045.


Journal Articles on this Report : 15 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
Type Citation Sub Project Document Sources
Journal Article Araujo JA, Barajas B, Kleinman M, Wang X, Bennett BJ, Gong KW, Navab M, Harkema J, Sioutas C, Lusis AJ, Nel AE. Ambient particulate pollutants in the ultrafine range promote early atherosclerosis and systemic oxidative stress. Circulation Research 2008;102(5):589-596. R832413 (2008)
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  • Journal Article Biswas S, Verma V, Schauer JJ, Cassee FR, Cho AK, Sioutas C. Oxidative potential of semi-volatile and non volatile particulate matter (PM) from heavy-duty vehicles retrofitted with emission control technologies. Environmental Science & Technology 2009;43(10):3905-3912. R832413 (2009)
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  • Journal Article 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(1):40-47. R832413C003 (2010)
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  • Journal Article Daher N, Ning Z, Cho AK, Shafer M, Schauer JJ, Sioutas C. Comparison of the chemical and oxidative characteristics of particulate matter (PM) collected by different methods:filters, impactors, and biosamplers. Aerosol Science and Technology 2011;45(11):1294-1304. R832413 (Final)
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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 Eiguren-Fernandez A, Miguel AH, Di Stefano E, Schmitz DA, Cho AK, Thurairatnam S, Avol EL, Froines JR. Atmospheric distribution of gas-and particle-phase quinones in Southern California. Aerosol Science and Technology 2008;42(10):854-861. R832413 (2008)
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  • Journal Article Eiguren-Fernandez A, Shinyashiki M, Schmitz DA, DiStefano E, Hinds W, Kumagai Y, Cho AK, Froines JR. Redox and electrophilic properties of vapor-and particle-phase components of ambient aerosols. Environmental Research 2010;110(3):207-212. R832413 (Final)
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  • Journal Article Hu S, Polidori A, Arhami M, Shafer MM, Schauer JJ, Cho A, Sioutas C. Redox activity and chemical speciation of size fractioned PM in the communities of the Los Angeles-Long Beach harbor. Atmospheric Chemistry and Physics 2008;8(21):6439-6451. R832413 (2008)
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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 Iwamoto N, Nishiyama A, Eiguren-Fernandez A, Hinds W, Kumagai Y, Froines JR, Cho AK, Shinyashiki M. Biochemical and cellular effects of electrophiles present in ambient air samples. Atmospheric Environment 2010;44(12):1483-1489. R832413 (Final)
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  • Journal Article Lin P, Yu JZ. Generation of reactive oxygen species mediated by humic-like substances in atmospheric aerosols. Environmental Science & Technology 2011;45(24):10362-10368. R832413C003 (Final)
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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 Shinyashiki M, Eiguren-Fernandez A, Schmitz DA, Di Stefano E, Li N, Linak WP, Cho S-H, Froines JR, Cho AK. Electrophilic and redox properties of diesel exhaust particles. Environmental Research 2009;109(3):239-244. R832413 (2008)
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  • Journal Article Verma V, Ning Z, Cho AK, Schauer JJ, Shafer MM, Sioutas C. Redox activity of urban quasi-ultrafine particles from primary and secondary sources. Atmospheric Environment 2009;43(40):6360-6368. R832413 (Final)
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  • Supplemental Keywords:

    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
  • 2008 Progress Report
  • 2009 Progress Report
  • 2010 Progress Report
  • 2011

  • 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