Gilmour et al. et al. 2004). Silica gel chromatography was used to fractionate organic DEP extracts into aliphatic, aromatic, and polar compounds, which were enriched for N-alkenes, PAHs, and quinones, respectively (Alsberg et al. 1985; Li et al. 2000, 2004). Among these, the polar compounds are the most potent in par- ticipating in redox cycling reactions as deter- mined by the interactions with the thiol derivative dithiothreitol (DTT). There is an excellent correlation between the results of the DTT assay and the ability of the various DEP fractions to induce oxidative stress in tissue culture macrophages and epithelial cells (Li et al. 2000, 2003b, 2004). Moreover, the same chemical groups are present in ETS, which may contribute to allergic airway inﬂammation in a fashion similar to DEPs. Although ROS is generally accepted as playing a role in asthma, demonstration of this in animals and humans involves mostly in- direct evidence from blood, bronchoalveolar lavage ﬂuid, and exhaled air samples. Although direct measurement of ROS production in the respiratory tract is difﬁcult, it is possible to use the biological impact of ROS generation in studying the effect of these radicals. When the production of ROS exceeds the ability of the affected tissue to neutralize the effects of the radicals, depletion in intracellular glutathione can result in a state of oxidative stress (Li et al. 2003a, 2003b). To develop new biomarkers for oxidative stress, several research groups used a proteomics display of newly induced oxidative stress proteins or posttranslationally GM-CSF trssse SCaOtDalase pTGr3Nleu8FccMeroAepcPctoKoerαrtpi1tcoorid2 ve Heme oxygenase-1 iNOS tia GAPDH etc. d Metallothionein ix etc. O Mitochondrial fumarate hydratase VDAC-1 FADD protein etc. Tier 1 Normal Antioxidant defense Tier 2 Inflammation Tier 3 Toxicity Cell response Figure 1. Hierarchical oxidative stress model in response to DEP exposure: proteome analysis of oxidative stress proteins shows a hierarchical response. Incremental doses of organic DEP extracts induce a series of incremental cellular responses that include increased antioxidant offense, inﬂammation, and cytotoxicity. At a lower level of oxidative stress (tier 1), antioxidant enzymes are induced to restore cellular redox homeostasis. At an intermediate level of oxidative stress (tier 2), newly expressed proteins often exhibit proinflam- matory activity. At a high level of oxidative stress (tier 3), perturbation of the mitochondrial permeabil- ity transition pore and disruption of electron trans- fer result in cellular apoptosis or necrosis. Abbreviations: FADD, Fas-associating protein with death domain; GM-CSF, granulocyte-macrophage colony-stimu- lating factor; iNOS, inducible nitric oxide synthase; SOD, superoxide dismutase; TNF, tumor necrosis factor; VDAC-1, voltage-dependent anion channel 1. 630 modiﬁed proteins in PM target cell types such as macrophages and epithelial cells (Wang et al. 2005; Xiao et al. 2003, 2005) Upon exposure to prooxidative organic chemical components from DEPs, these cells show three tiers of oxidative stress, which are predic- tive of a possible in vivo hierarchical oxidative stress response (Figure 1) (Li et al. 2003a; Xiao et al. 2003). This model posits that as the level of oxidative stress increases, there is a transition from protective to injurious effects. Not only do these biological outcomes provide possible biomarkers of oxidative stress, but this model could also provide clues about susceptible human subjects. In the ﬁrst tier of oxidative stress, epithe- lial cells and macrophages respond by increas- ing expression of antioxidant and phase II enzymes (e.g., superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, γ-glutamate cysteine ligase, glutathione S-transferase, NADPH quinone oxidoreduc- tase, heme oxygenase 1) via a genetic response pathway that acquires transcriptional activa- tion of the antioxidant response element (ARE) by the transcription factor nuclear regulatory factor 2 (Nrf2) (Li et al. 2004). The Nrf2–ARE pathway protects against the inju- rious and proinﬂammatory effects of PM, and it has been proposed that a weakening of antioxidant defense (e.g., by the polymor- phism of these genes) could determine which human subsets are susceptible to PM effects (Fryer et al. 2000). This notion is supported by recent data showing an increased asthma frequency in people with a polymorphism in the glutathione S-transferase who are more prone to increased IgE production in response to an intranasal co-challenge with allergen and DEPs (Gilliland et al. 2004). Similarly, there is evidence in animal asthma models that over- expression of heme oxygenase 1, a phase II enzyme with potent antioxidant effects, leads to a blunting of AHR and airway inﬂamma- tion (Hisada et al. 2000). In the second tier of oxidative stress, the activation of several intracellular signaling cas- cades could lead to transcriptional activation of proinflammatory genes (Xiao et al. 2003). Phosphoproteome analysis and anti-phospho- peptide immunoblotting demonstrate the acti- vation of three major mitogen-activated protein (MAP) kinase cascades in epithelial cells and macrophages by DEPs, crude DEP extracts, and aromatic and polar DEP fractions (Wang et al. 2005; Xiao et al. 2003). Activation of these pathways leads to tumor necrosis factor-α, interleukin (IL)-8, IL-6, and vascular endothe- lial growth factor production that could be inhibited by MAP kinase inhibitors (Wang et al. 2005). It is noteworthy that the induction of AHR and airway inflammation can be blocked in a murine asthma model by employ- ing a small-molecule inhibitor that interferes in the transcriptional activity of AP-1 (activator protein 1) proteins. There is good evidence that the above cytokines and chemokines affect air- way inflammation and asthma, thereby con- tributing to the effects of the classical TH2 cytokines such as IL-4, IL-5, and IL-13. Nasal challenge studies with DEPs demonstrated that PM enhances TH2 cytokine production in atopic people during co-challenge with an aller- gen. These cytokine effects could form the basis for the adjuvant effects of DEPs on allergic inﬂammation in addition to the possibility that these particles affect the function of antigen presenting cells (Nel et al. 1998). The third tier of oxidative stress involves mitochondrial perturbation, which can lead to apoptosis, apoptosis–necrosis, and superoxide generation (Hiura et al. 1999). In this regard, it is relevant that ambient ultraﬁne PM (PM < 0.1 µm) lodges in and induces structural dam- age in mitochondria (Li et al. 2003b). Although the clinical relevance of mitochondrial damage is uncertain, it is relevant that ultraﬁne PM col- lected in the Los Angeles basin has a higher content of redox-cycling organic compounds that are more prone to generate ROS in the DTT assay than does PM2.5 and PM10 (Li et al. 2003b). DEPs contribute signiﬁcantly to the ultraﬁne PM load in an urban area. Although there is ample epidemiologic evidence that PM10 and PM2.5 play a role in asthma, there is a paucity of data on the role of ultraﬁne PM in asthma. However, on the basis of their small size, large surface area, high numbers, rich con- tent of redox cycling chemicals, and high depo- sition efficiency, ultrafine PM may be particularly prone to induce airway inﬂamma- tion and AHR (Penttinen et al. 2001). Since vehicular emissions have a signiﬁcant output of ultraﬁne particles, it is relevant, therefore, that a number of studies have shown a link between trafﬁc density and asthma exacerbation in inner city populations (Brauer et al. 2002). In summary, there is growing evidence that PM-induced oxidative stress may be responsi- ble for generating airway inflammation and AHR that are both markers and possibly pre- cursors for the development of asthma. In addition, there is the realization that not all oxidative stress responses are injurious but also include a protective lower level of oxidative stress (tier 1 response; Figure 1) that could form the basis of disease susceptibility. Impact of Global Warming and Climate Change on Aeroallergens Climate and allergen load. It is generally thought that for atopic asthma or allergic dis- ease to develop, both genetic predisposition and allergen exposure are required. Therefore, it is important to understand how impending climate change will affect the aeroallergens that elicit disease onset and precipitate symptoms. VOLUME 114 | NUMBER 4 | April 2006 • Environmental Health Perspectives