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Single exposure to near roadway particulate matter leads to confined inflammatory and defense responses: possible role of metals Michal Pardo, Martin M. Shafer, Assaf Rudich, James J. Schauer, and Yinon Rudich Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b01449 • Publication Date (Web): 29 Jun 2015 Downloaded from http://pubs.acs.org on July 2, 2015
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Single exposure to near roadway particulate matter leads to confined
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inflammatory and defense responses: possible role of metals
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Michal Pardo,1 Martin M. Shafer,2 Assaf Rudich,3 James J. Schauer,2 and Yinon Rudich1*
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1
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76100, Israel. E-mail:
[email protected]
Department of Earth and Planetary Sciences, Weizmann Institute of Science, Rehovot
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2
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Madison, WI, USA
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3
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National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-
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Sheva, Israel
Environmental Chemistry and Technology Program, University of Wisconsin-Madison,
Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, and the
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Key words: Roadway metals, Air Pollution, Oxidative Stress, Nrf2, Inflammation, Intra-
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tracheal installation
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ABSTRACT
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Inhalation of traffic-associated atmospheric particulate matter (PM2.5) is recognized as a
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significant health risk. In this study we focused on a single ("sub-clinical response") exposure
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to water-soluble extracts from PM collected at a roadside site in a major European city, to
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elucidate potential components that drive pulmonary inflammatory, oxidative and defense
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mechanisms, and their systemic impacts. Intra-tracheal instillation (IT) of the aqueous
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extracts induced a 24h inflammatory response characterized by increased broncho-alveolar
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lavage fluid (BALF) cells and cytokines (IL-6 and TNF-α), increased reactive oxygen species
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production, but insignificant lipids and proteins oxidation adducts in mice' lungs. This local
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response was largely self-resolved by 48h, suggesting that it could represent a sub-clinical
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response to everyday-level exposure. Removal of soluble metals by chelation markedly
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diminished the pulmonary PM-mediated response. An artificial metal solution (MS)
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recapitulated the PM extract response. The self-resolving nature of the response is associated
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with activating defense mechanisms (increased levels of catalase and glutathione peroxidase
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expression), observed with both PM extract and MS. In conclusion, metals present in PM
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collected near roadways are largely responsible for the observed transient local pulmonary
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inflammation and oxidative stress. Simultaneous activation of the antioxidant defense
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response may protect against oxidative damage.
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INTRODUCTION:
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Outdoor air pollution by fine atmospheric particles is a significant public health risk
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that contributes to over 3.2 million premature deaths annually worldwide, thus placing
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outdoor air pollution among the top 10 global mortality risks.1 The effect of near roadway air
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pollution on human health is especially significant in major cities, with respiratory and
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cardiovascular diseases being mostly implicated in response to continued/repeated exposure
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to air pollution.2-7 In an effort to limit roadway-related air pollution, the common regulations
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have focused on reducing tailpipe emissions. Among the various particulate matter (PM)
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constituents,8 some metals9, 10 are potentially cytotoxic and can contribute to organ and tissue
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damage and injury.3, 11,
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sources of these metals it is expected that their roadway emissions are largely associated with
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re-suspended road dust that contains brake wear, tire wear, and crustal elements.13-16
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Recently, Verma et al. showed that all major components of fine PM, including metals,
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contribute to reactive oxygen species (ROS) generation, and that exposure to aerosol
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components can catalyze production of oxidants in vivo.17 Consistently, PM0.25-2.5 samples
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collected in Beirut near a freeway contained high concentrations of metals and trace elements
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(including Mn, Cr, Cu, and Ba) that correlated with in vitro ROS-activity in macrophage
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cells.18 Several toxicological studies established associations between various coal and
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residual oil fly ash (ROFA; a transition metal-dominated sample) metals and the PM-induced
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inflammation in lung tissue and broncho-alveolar lavage fluid (BALF).19-21 Metals such as
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Pb, Cr, V, Fe, Cu, Mn, Sr and Ba, were measured at relatively high levels near roadway
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samples.15,
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presents a major challenge in identifying specific components that are most biologically
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active and/or leading to pathogenesis of disease. Identifying these components is essential for
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appropriate mitigation policies that could reduce their public health impact.
22, 23, 24
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Yet, since tailpipe emissions from mobile sources are not major
The heterogeneity of chemical species and different roadway sources
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Oxidative damage is caused when the production of ROS exceeds their removal by
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antioxidant defense mechanisms, and different cellular constituents are consequently
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oxidized. Transition metals present in PM can induce ROS production, and the ensuing
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oxidative stress was proposed to constitute a major mechanism mediating PM -induced health
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impacts.6 Recent studies reported that PM-derived water-soluble transition metals (e.g. Fe,
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Ni, Cu, Cr, Mn, Zn and V) significantly correlate with the oxidative potential of airborne PM
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across different urban areas and particle size ranges.25,
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oxidative stress by up-regulating a distinct array of cyto-protective genes responsible for the
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cells’ antioxidant and detoxification capacity.27 Some of these genes act to maintain cellular
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reduced glutathione (GSH) levels and high reduced/oxidized glutathione (GSH/GSSG)
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content. A master regulator of this anti-oxidant response is the transcription factor NF-E2-
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related factor-2 (Nrf2).27 Indeed, several studies demonstrated that Nrf2 is activated in vivo
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and in vitro after exposure to diesel exhaust particles, PM, or to engineered nano-particles
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(NP).28, 29 Moreover, the degree to which Nrf2-dependent antioxidant response functionally
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meets the oxidative stress challenge posed by PM/NPs likely determines cell fate.29
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Cells or tissues can respond to
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In this study we aimed at identifying the in vivo role PM-derived transition metals
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from collected roadside PM in mediating pulmonary inflammatory and oxidative stress
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response, as well as the Nrf2-related antioxidant/detoxification defense mechanism. Metals
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present in the water-soluble extracts from collected roadway particles were either chelated
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from the extracts, or in complementary experiments, a water metal solution containing the
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same composition and concentrations of the PM-derived metals was used, and the effect on
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pulmonary and systemic inflammation and oxidant stress and defense were assessed. We
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used a single, low-dose exposure by intra-tracheal instillation (IT) to elicit a transient
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inflammatory/oxidative response rather than a toxic/full-blown disease-promoting exposure
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level, to model pulmonary response to common levels of daily exposure to roadway PM in an
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urban environment.
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MATERIALS AND METHODS
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PM collection and water extract preparation. Samples were collected from a roadside
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monitoring site in central London (Marylebone Road near Baker Street) in early July 2012. A
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Hi-Vol sampler (Tisch (TE-230) Hi-Vol Environmental Impactor Sampler), configured for
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three particle size cuts (10 µm) and fitted with pre-cleaned mixed-
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cellulose ester (MCE) substrates, was deployed to obtain two time-segregated samples.
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Samplers were run continuously for 3-4 days at nominal flow rates of 1.2 m3 min-1, sampling
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5000 m3 of air, and collecting from 50-300 mg of PM onto the MCE filter media.
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Sub-sections of the MCE filter-collected PM3 were digested for total metals
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(microwave aided mixed-acid digestion) and extracted with high-purity Milli-Q (18 mΩ)
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water using an initial sonication period of 15 minutes (min) followed by 16h of continuous
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agitation at room temperature in dark conditions and then finally another 15 min sonication.
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At the end of the extraction period, aliquots of the suspension were removed and distributed
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for various assays and analyses (PM extract), and the remaining suspension volume
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centrifuged (6600 rpm for approximately 1 min) and then filtered through 0.22 µm
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polypropylene syringe filters. Soon after filtration, an aliquot of the filtered sample was
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processed through a miniature column of carefully pre-cleaned Chelex 100 resin (Bio-Rad
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#143-2832, imino-diacetate in sodium form) to remove/isolate Chelex-labile (truly soluble
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ions and “loosely” bound) metals (Chlx). The Chelex treatments were performed with 0.2g of
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Chelex loaded into 1.5 mL polypropylene SPE reservoirs at a sample flow rate of 1 mL/min.
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Method blanks replicated the processing procedure (Control). The extracts and digests of the
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PM were subjected to a broad range of characterization tools, including: total and water-
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soluble elements [ICPMS (SF-ICPMS)]; soluble ions (K+, Na+, NH4+, SO42-, NO3-, Cl-) by -6ACS Paragon Plus Environment
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IC; soluble organic carbon; and several toxicity assays. The TOTAL elemental mass
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extracted from the London PM (microwave-aided acid digestion), was normalized to the PM
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mass to enable a direct comparison with similarly normalized data from the unfiltered water
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extract data. Using a chemical species mass model, the total mass that these elements
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contribute to the total PM was calculated. The filters show more than 80% recovery of the
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target metals and inorganic species.
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Metal solution (MS) preparation. To investigate the influence of metals on the bioassays, a
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series of pure metal solutions were prepared to mimic the concentration of metals in the
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extracts of the PM samples. The specific metal mixtures were: (a) major cations/anions to
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ensure that we captured the bulk metals and ion-strength [Ca, Mg, Na, K, Ba, Sr, S, P], (b)
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major and minor cationic/anionic redox-active transition metals [Fe, Mn, Cu, V, Ni, Cr], (c)
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major non-redox active metals [Zn, Pb, Al], and (d) several minor elements of significance in
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roadway emissions [Y, Ce, Pt and Pd]. The metal mixture solutions as defined in (a)–(d) were
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prepared from high-purity individual element NIST-traceable stock standards (High Purity
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Standards, Charleston SC). Metal concentrations in the 4 solutions were such that after a 10-
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fold dilution of the solution the individual metal concentrations in the exposure solution
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would be at the target levels. We used the cations solution (a) and the redox active metals (b)
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MSs were prepared in acid-washed LDPE bottles using high-purity (18.2 MΩ) water–
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sufficient acidity remained from the original stock solutions to maintain metal stability. The
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exposure matrix provided adequate buffering to sustain optimum bioassay pH. See Table 1
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for concentrations in the metals solution.
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Mice treatments. Male C57BL/6 (7-8 weeks) mice were housed under standard light/dark
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conditions and were given access to food and water ad libitum. Experiments were approved
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by the Animal Care and Use Committee of the Weizmann institute of Science. Mice were
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randomly divided into groups (minimum, n=6), where different water extracts and solutions
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were used: water extract from urban London PM sampling (“PM extract”); water extract from
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urban London PM sampling after chelation of soluble metals (“Chlx”) (non-specific sorption
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of hydrophobic organic carbon to the resin backbone may occur, however the loss is minimal
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the London water extract (“MS”). Pure metal solutions containing major cations: (Ca, Mg,
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Na, K, Ba, Sr, S and P) (“Cations”) and redox active metals (V, Ni, Cu, Fe, Mn, and Cr)
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(“Redox Active”). Control animals were exposed to blank extracts suspension in a
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corresponding manner, another control group exposed mice to the PM extract using
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intraperitoneal injection (i.p) (Supporting Information (SI), Table S1.). Prior to the mice
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exposure, salts glucose medium, consisting of 500mM HEPES, 1MNaCl, 50mM KCl, 20mM
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CaCl2 and 50mM dextrose PH 7.2, were added to sub-samples of the PM extracts, then pH
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was 7.5. After i.p anaesthesia with ketamine/xylazin (10mg/kg and 5mg/kg body weight,
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respectively), each mouse was placed on an inclined plastic platform; the dosing of
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particulate suspension was performed while the tongue was pulled out with forceps to prevent
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the mouse from swallowing the sample. The extract dose was delivered onto the vocal cords
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and afterwards, the nostrils were covered, forcing the mouse to inspire the instilled particle
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suspension. Each mouse received a single dose of 50µl PM water extract corresponding to
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10µg PM2.5. Twenty-four hours (h) or 48h after instillation, all mice recovered the procedure
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with no apparent health consequences. Then, mice were anesthetized again by i.p
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ketamine/xylazin (20mg/kg and 10mg/kg body weight, respectively); blood was taken from
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the retro-orbital vein for serum-cytokine analysis. Whole-body perfusion with PBS was
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performed, the lungs and tracheas were exposed by dissection, tracheal cannula was inserted.
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Lungs were lavaged with PBS solution twice. Cells were separated from the BALF by
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centrifugation (500 × g, 5 min). The supernatant was removed and the cells were re-
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suspended in 100 µl of sterile saline for microscopic counting using hemocytometer and
); a metal solution mixture containing metals at concentrations similar to those present in
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Turk’s solution (crystal violet and 6% acetic acid) exclusion method.
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Tissue preparation. Immediately after taking the BALF, the left lung was removed and
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divided to several sections which were snap-frozen in liquid nitrogen for future analysis. The
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remaining lung tissue (50-70mg) was rinsed with PBS and placed in a buffer containing
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0.25M sucrose, 1mM EDTA, and 5mM HEPES, pH 7.4, followed by mincing the tissue
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using scissors and pestle. The resulting homogenate was centrifuge 14,000g for 5 min to
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remove cell debris.31 The supernatant was immediately analyzed for ROS and subsequent
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protein determination using the Bradford method.32
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ROS analysis. ROS were detected using 2’,7’–dichlorofluorescin diacetate (DCFDA)
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detection kit (Abcam, UK) on fresh BAL cells and fresh lung tissue according to manufacture
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instructions. Briefly, fresh cells and tissue homogenates were incubated with DCFDA for 30
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min at 37 °C, fluorescence was measured with maximum excitation and emission spectra of
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495 and 529 nm respectively in a microplate reader. Hydrogen peroxide (H2O2) and Tert-
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Butyl hydrogen peroxide (TBHP) were used as positive controls. Calibration curves were
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performed with H2O2 on lung Tissue and BAL cells respectively (SI, Figure S1A).
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Immunoassay Measurements for Cytokines Secretion. Mouse ELISA kits (eBioscience,
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Affymetrix, CA) were used according to the manufacture’s instructions to measure IL-6, and
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TNF-α secretion in BALF and in the serum. Concentrations of IL-6, and TNF-α were
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calculated from a calibration curve with known (supplied) standards. The absorbance of the
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samples was measured at 450 and 570 nm by a microplate reader.
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Lipid peroxidation. The extent of lipid oxidation in lung tissue was evaluated using the
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thiobarbituric acid (TBA) method,33 producing a pink pigment. The lung homogenate was
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mixed 1:1 with 50% trichloroacetic acid to precipitate the protein. After centrifugation, the
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supernatant was reacted with TBA (280mg in 100 ml) in a boiling water bath for 10min.
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After cooling, the absorbance at 532 nm using was measure using a spectrophotometer. The
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values obtained were compared with a series of MDA tetrabutylammonium salt standard
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curve (Sigma-Aldrich, St. Louis, MO, USA) (SI, Figure S1B).
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Protein carbonyl content. Protein carbonyl content, a general indicator for protein
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oxidation, in lung tissue homogenates was measured using protein carbonyl calorimetric
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assay (Cayman chemicals, Ann Arbor, Michigan, USA) according to manufacture
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instructions. Briefly, the reaction between 2,4, dinitrophenyl hydrazine (DNPH) and protein
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carbonyl forming a Schiff base and produces a corresponding hydrazone was measured. The
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amount of protein-hydrazone was quantified at 370 nm using a spectrophotometer. Protein
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carbonyl content was calculated using the extinction coefficient of 22,000 M− 1 cm− 1 and
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expressed in nmol/mg protein.
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Measurements of total GSH. Total glutathione (oxidized + reduced; GSSG + GSH) in the
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lung tissues was determined using a glutathione assay kit (Sigma-Aldrich). Briefly, snap
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frozen lung tissues were minced and suspended in 5% 5-sulfosalicylic acid and incubated in a
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reaction mixture containing 95mM potassium phosphate buffer, 0.95mM EDTA, 48µM
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NADPH, 0.031mg/ml DTNB, 0.115units/ml glutathione reductase, and 0.24% 5-
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sulfosalicylic acid. The absorbance was measured at 412 nm using a microplate reader with
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kinetic read. The standard curve was obtained from absorbance of the diluted commercial
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GSH.
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Gene expression; RNA isolation, reverse transcription (RT), and real-time PCR. Total
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RNA was extracted from lung tissue, using RNeasy RNA kit (QIAGENE, Hilden, Germany).
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RT and real-time PCR were performed for quantification of mRNA expression using the
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SYBR Green PCR mix (Applied Biosystems, Foster City, CA, USA) in StepOnePlus RT
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PCR instrument. Total RNA (0.5 µg) was reverse-transcribed to cDNA using random
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30 s and 60 °C for 30 s and extension at 72 °C for 30 s. β-actin and 18S mRNA were used as
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endogenous controls. Melt-curve analysis showed a single peak for the primer sets of genes,
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indicating no primer dimer formation. RNA samples were used as non-RT controls to exclude
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interference by genomic DNA contamination. Slopes were – 3.4 for both target and
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endogenous control genes. cDNA dilutions for the samples were set such that ∆CT between
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the target and the endogenous control genes was no more than five cycles. Primers were
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purchased from Sigma-Aldrich. (Rehovot, Israel) and are described in the following table (SI,
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Table S2).
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Statistical analysis. Results are expressed as means ± standard errors of the mean (SEM).
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One-way ANOVA was used in multivariable analyses. Statistical evaluation was performed
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with OriginLab (Data Analysis and Graphing Software, Northampton, MA, USA).
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Differences were considered significant at probability level p < 0.05 using the Fisher
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protected least-significant difference method.
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RESULTS AND DISCUSSION
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In this study we used 4 solutions: 1. “control”: Method blanks that replicated the entire
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procedure, without PM2.5 extracts; “PM extract”: Water extracts from PM collected in a
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near roadway station in London. 3. “Chlx”: Chelated PM extracts, where the water extracts
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passed through a Chelex column to remove soluble metals. 4. “MS”: an artificial metal
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solution with similar metal concentrations to those present in the “PM extract” solution.
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Detailed description of the preparation is given in the Methods section above.
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Inflammatory responses in BALF
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Cell count in BALF was used to estimate the pulmonary inflammatory response to a
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single, low-dose, intra-tracheal instillation of extracts of water-soluble material from - 11 ACS Paragon Plus Environment
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collected near-roadway PM (“PM extract”). After a single low dose exposure of mice to
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urban PM water extracts, the total cell number in the BALF increased significantly after 24h,
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consistent with previous studies.24,
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specific systemic inflammatory response, as the same dose of PM extract administered intra-
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peritoneally (i.p) failed to elicit a similar response (SI, Table S1). To test the contribution of
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metals to this biological response to PM extract, two complementary experiments were
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performed: i. Metals were removed from the PM-water extract by filtration and chelation on
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a column (“Chlx” solution), and the response was compared to that of the “PM extract”
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solution. ii. An artificial aqueous metal solution (“MS” solution) containing only the metals,
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with the same content and concentration as the PM extract itself (Table 1) was prepared to
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test if it could recapitulate the response to the “PM extract” solution. The Chlx column indeed
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greatly depleted the PM water extract from metals by >95% (Table 1). Exposure to the metal-
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depleted solution induced a markedly lower increase in BALF cell content 24h following IT
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administration (Figure 1). Conversely, the MS solution induced a significantly increased
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BALF cellular content that was not statistically different from the response after exposure to
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the PM extract (Figure 1).
34, 35
This response in the BALF did not reflect a non-
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To determine if the 24h time point represents the initiation of a fulminant pulmonary
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inflammatory response, BALF were obtained also 48h after extract administration. While
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after 24h BALF cell counts were >3-fold higher in response to PM extracts compared to
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control solution, at 48h only a ~50% increase in cell number was observed (results not
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shown), suggesting significant and near-complete resolution of this inflammatory response of
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the lungs by 48h post instillation.
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To better characterize the inflammatory response observed in response to PM extract
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and MS exposure, and to assess its systemic impact, TNF-α and IL-6 levels were measured in
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several fold in BALF in response to either PM water extract or to the artificial MS solution
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compared to control (Figure 2A, B). Moreover, removal of metals by chelation fully
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abrogated this pro-inflammatory response to the PM extract. This inflammatory response was
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fully resolved by 48h post installation (data not shown). The onset of pulmonary
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inflammation evident here by the increased cytokines levels and cell numbers in the BALF is
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consistent with a previous study,36 and with previous observations of increased neutrophil
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numbers in BALF after exposure to road tunnel dust,37 urban air5 and mineral dust.3
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Furthermore, our results strongly implicate water-soluble metals in this response, and indeed,
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metals were suggested as major contributors to the inflammatory response to PM2.5
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samples.3, 35
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It has been suggested that cytokines produced in response to PM inhalation in the lung
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may enter the blood stream and activate inflammatory response in remote organs (e.g heart,
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adipose tissue, and muscle).38,
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evaluated to assess the systemic impact of this low-level PM extract exposure. Serum TNF-α
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and IL-6 levels remained unaltered in the 24h and 48h post-exposure to the PM water extracts
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(SI, Figure S2).
39
Therefore, levels of TNF-α and IL-6 in serum were also
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Overall, the results described so far suggest that inhalation of PM water extract at the
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dose used induce a transient, self-resolving inflammatory response without gross systemic
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inflammatory impact. Consistently, BALF cell number and cytokines levels showed
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approximately the same magnitude of increase (about 3.5 fold increase for the cell count and
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IL-6, and 2.5 for TNF-α), implying that increased cytokines content can mainly be attributed
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to the higher cell recruitment into the lungs, with immune activation playing a minor role.
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This further supports the notion that the exposure level used reflects a low-dose, transient,
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local reaction in the lungs that minimally manifests systemically.
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ROS levels were measured in fresh tissue homogenates using 2’,7’–dichlorofluorescin
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diacetate (DCFH-DA) probe where H2O2 and Tert-Butyl Hydrogen Peroxide (TBHP) were
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used as positive controls. A major possible limitation of this assay is that the probe can be
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oxidized by a number of free radicals and is not ROS-specific. DCFH competes with
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antioxidants as well as other cell macromolecules and therefore serves only as an estimate of
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ROS generation. Nevertheless, in this experiment we show that 24h following exposure to the
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PM water extract and to the artificial MS induced ROS generation, while the control and
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Chlx samples did not (Figure 3A). After 48h, ROS levels were lower than after 24h exposure
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and not significantly higher than those of the control (results not shown). These observations
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are in agreement with studies showing that PM-associated metals can increase oxidative
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stress and inflammation, thereby contributing to adverse health effects of air pollution.6, 9, 31,
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36, 40
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H2O2 in a surrogate lung fluid.41
Moreover, transition metals such as Cu and quinones from PM produced high levels of
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When ROS production exceeds the capacity to neutralize them, oxidatively-modified
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diverse cellular macromolecules, including nucleic acids, proteins and lipids accumulate,
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indicating oxidative damage.42 To assess whether the inflammatory and oxidant effect of PM
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extract and MS resulted in measurable oxidant damage, lipid peroxidation and protein
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carbonyl products were measured. Neither the PM water extract, nor the artificial MS,
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changed lung tissue malondealdehyde (MDA) levels, 24 and 48h after installation (Figure 3B,
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the 48h results are not shown). Similarly, the protein carbonyl content in the lung tissue did
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not change following exposure to PM extracts (Figure 3C). The lipid peroxidation finding
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differs from the prominent increase in peroxidative damage observed following exposure of
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mice' lungs to low doses (15 µg) urban PM collected in Buenos Aires,5 or exposure of rat
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lungs to coal dust PM10,43 and in RAW 264.7 cells exposed to welding fumes (which
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contains a complex mix of metal oxide particles).44 Collectively, these data indicate that - 14 ACS Paragon Plus Environment
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exposure to PM containing high concentrations of metals induces lung lipids damage. Our
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data suggests that the levels of metals in the soluble fraction of the roadside PM we used
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(source: London) were too low (10µg dose) to induce demonstrable pulmonary lipid
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peroxidation toxicity response upon exposure to a single dose, despite mounting a measurable
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inflammatory and ROS production response. This suggests that PM extract or MS at the
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conditions used did not exceed the antioxidant/detoxifying capacity of the tissue.
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Nrf2 phase II antioxidant defense
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We next explored whether the apparent compensation against tissue damage indeed
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engaged activation of defense mechanisms. To this end, we assessed phase II detoxifying and
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antioxidant enzymes - classical targets of Nrf2, the principal regulator of this major cellular
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antioxidation mechanism, acting by binding the antioxidant response element (ARE) in its
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target genes' promoters.45 Indeed, PM water extract and MS produced a significant (2.5-fold)
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rise in the stress-induced protein heme oxygenase-1 (HO-1) (Figure 4A). Antioxidant genes
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such as catalase and glutathione peroxidase (GPx) also increased by 2.5 fold (each,
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respectively) (Figure 4B, and C, respectively) with exposure to PM extract and MS at 24h. In
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contrast, after removal of the metals by chelation (Chlx), the levels of these antioxidant genes
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were similar to the control (Figure 4A, B, C). An additional, major cellular antioxidant
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regulated by Nrf2 is the tripeptide GSH. We observed that the PM water extract and the MS
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increased GSH levels, while the chelated solution induced a lower effect, which was not
341
statistically significant. No significant effects in the antioxidant genes and in GSH compared
342
to control were observed 48h following exposure (results not shown), further supporting the
343
transient, self-resolving PM-induced inflammatory/oxidative response. Therefore, it is
344
possible that the glutathione system, along with antioxidant and detoxification enzymes may
345
counteract ROS generation induced by PM extract and by the MS, thereby maintaining tissue
346
homeostasis and avoiding oxidative pulmonary damage. - 15 ACS Paragon Plus Environment
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These observations are in accordance with our previous report demonstrating that
348
some engineered nanoparticles can activate the antioxidant response via Nrf2-ARE -mediated
349
Phase II detoxification genes in human bronchial epithelial NL-20 cells.29 These results are in
350
agreement with experiments showing Nrf2-mediated protection against TiO2 NPs that
351
induced pulmonary damages in mice,46 and with Nrf2 deficient mice that exhibited reduced
352
levels of GSH and HO-1 expression in lung tissue in response to cigarette smoke compared to
353
mice with over-expressed Nrf2.47 In contrast, it was found that exposure of rats to PM2.5 (7.5
354
mg/kg) significantly increased lipid peroxidation levels in the heart, liver, and lungs, depleted
355
GSH, decreased catalase, and GPx activities in the lungs, liver, and kidneys.48 These
356
differences may be attributed to the different dose of the activating substances in the
357
administered PM. Our results suggest that the exposure in the present study leads to a low-
358
grade transient inflammatory response that does not induce severe oxidative damage,
359
potentially by invoking protective mechanisms. Our findings also imply that Nrf2 may
360
mediate a central adaptive intracellular response to metals-containing PM, preventing
361
oxidative stress in the mouse lung.
362
We can relate the inflammation and oxidative stress induced by water extracts of
363
roadside PM to the presence of metals. While chelation of the metals from the extract
364
significantly attenuated the inflammatory/oxidative response to PM extract to levels
365
comparable to control, the response to the artificial MS reproduced most of that response in
366
all parameters measured. The possible effect of organic compounds and biological
367
components cannot be ignored; however, the results of the metal chelation and MS suggest
368
that in this case they likely account for a small fraction of the effect.
369
Several specific active metals (Cu, Fe, Mn, V, Ni, and Cr) were found in high
370
quantities in London roadside pollution (Table 1). To verify the possible effects of these
371
redox-active metals at concentrations relevant to roadside PM, we again prepared solutions - 16 ACS Paragon Plus Environment
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that contained these metals at the relevant concentrations and compared their oxidative and
373
inflammatory potential to the MS and to another solution that contained other cations that are
374
also found in the PM extract (Ca, Mg, Na, K, Ba, Sr, S, P, Table 1). The solution of the
375
redox-active metals indeed increased lung inflammation more than the cations solution
376
(expressed as percentage of the response to MS), as reflected by increase in cytokines (IL-6;
377
37% versus 1%, TNF-a; 24% versus 1% respectively) and BALF cell number (84% versus
378
49% respectively) (Table 2). The redox-active metals exposure resulted in both higher ROS
379
generation (73% versus 48% respectively) and higher induction of the Nrf2 phase II enzymes
380
(Table 2, The nominal values of each assay with the redox active and the cation groups were
381
statistically different in all parameters presented). These results are in line with previous
382
studies that have identified Cu, Fe, and Ni,
383
induce inflammation and oxidative stress using a variety of techniques, including the DTT 51,
384
52
22, 49
and Cr from PM
50
as important agents that
and DCFH-DA 18, 25 assays.
385
This study shows that exposure to PM extracts collected in a near roadway
386
environment can induce a transient oxidative stress and inflammation in mice' lungs, which is
387
largely attributable to the dissolved metals (such as Cu, Fe, Mn, V, Ni, and Cr) that are part
388
of roadway emissions.15,
389
chelation did not cause the same in vivo effects as the metal-containing PM extract. The
390
artificial metal solution recapitulated the response induced by PM extract, thus excluding a
391
major role for other cheletable (organic) material. It is suggested that the increase in the
392
antioxidant defense system, potentially regulated by Nrf2, protects against oxidative damage
393
by the metals. Jointly, this low-dose, compensated and short-lived response may elucidate the
394
sequence of events that occur in the respiratory system by normal daily-life exposure to
395
roadside-derived air pollution. Metals in re-suspended roadway PM are largely derived from
396
brake and tire wear, and re-suspension of road dust 14. These roadway sources are typically
23
This conclusion was confirmed by the observation that metal
- 17 ACS Paragon Plus Environment
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397
not a focus of emission controls measures, which are largely targeted at tailpipe emissions.
398
Future studies should unravel whether repeated low-dose exposure eventually results in de-
399
compensated biological response and accumulated damage that could explain air-pollution –
400
associated morbidity and mortality. In such case, regulating non-tailpipe –related pollution
401
sources must be considered
402
human health.
30
to alleviate the impact of traffic-associated air pollution on
403 404 405 406 407 408 409 410 411 412 413 414 415 416
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417
Figure 1. Increased cell number in broncho-alveolar lavage fluid of C57BL/6 mice
418
exposed to PM extract and MS
419
1200000
*
*
Cell Count (Cells/ml)
1000000
800000
600000
400000
200000
0
Control
PM extract
Chlx
MS
420 421 422
Total cell number in Broncho alveolar lung fluid (BALF) after 24 h of intra-tracheal (IT)
423
exposure to particulate suspension (10 µg) in C57BL/6 mice. Data represent means ± SE;
424
n=7-9 mice per group; * significantly higher at p < 0.05 than their controls (mice subjected to
425
IT with blank water extract).
426 427 428 429 430 431 432
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433
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Figure 2. TNF-α and IL-6 cytokines increased in BALF exposed to PM extract and MS
434
A.
B. 140
*
*
500
120 100
TNF-α α (Pg/ml)
IL-6 (Pg/ml)
*
*
400
300
200
80 60 40
100
20 0
0
Control
PM extract
Chlx
Control
MS
PM extract
Chlx
MS
435 436 437
(A) IL-6 and (B) TNF-α cytokines in BALF, after 24 h of IT exposure to particulate
438
suspension (10 µg) in C57BL/6 mice. Data represent means ± SE; n=7-9 mice per group; *
439
significantly higher at p < 0.05 than their controls.
440 441 442 443 444 445 446 447 448
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449
Figure 3. Increased ROS with no change in lipid peroxidation and protein carbonyl in
450
lungs of mice exposed to PM extracts or metal solution
451 B.
20
Carbonyl content (nmol/mg Protein)
A. 2.0
*
*
MDA (nmole/µ µ gr Protein)
ROS [DCF Fluorescence (A.U)]
10
1.5
1.0
0.5
8
6
4
2
0.0
0
Control
PM extract
Chlx
MS
C.
15
10
5
0
Control
PM extract
Chlx
MS
Control
PM extract
Chlx
452 453
(A) Tissue ROS was measured by H2DCF-DAdetection kit as described in material and
454
method section, values were calibrated to protein levels examined by Bradford method. (B)
455
Lipid peroxidation was measured in lung tissue homogenates and was calibrated to protein
456
levels examined by Bradford. (C) Protein carbonyl was measured in lung homogenates and
457
was calibrated to protein levels examined by Bradford. Data represent means ± SE; n=7-9
458
mice per group; * significantly higher at p < 0.05 than their controls.
459 460 461 462 463 464 465
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466
Figure 4. Nrf2- phase II protective pathway is increased after exposure to PM extract
467
and MS
4
*
* 3
2
1
0
Control
PM extract
Chlx
MS
B.
C. *
*
3
2
1
0
Control
PM extract
Chlx
MS
Relative quantification of Gpx expression (A.U)
A.
Relative quantification of catalase expression (A.U)
Relative quantification of HO-1 expression (A.U)
468
4.0
*
3.5
*
3.0 2.5 2.0 1.5 1.0 0.5 0.0
Control
PM extract
Chlx
MS
D. 3
GSH (Folds of control)
*
* *
2
1
0
Control
PM extract
Chlx
MS
469 470 471
Quantitative analysis of Nrf2-target genes was performed by RT and real-time PCR for
472
mRNA levels of (A) HO-1 (B) catalase and (C) GPx. Values are expressed as fold change of
473
gene expression compared to a calibrator (endogenous control, β-Actin). (D) GSH content
474
was determined in lung tissue homogenate. Data represent means ± SE; n=7-9 mice per
475
group; * significantly higher at p < 0.05 than their controls.
476 477 478
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479
Table 1. Metal concentration from filters sampled near roadway in urban London,
480
before and after metal Chelation and the concentrations in the metal solution
481
Element
PM extract (µg/Liter)
Chlx (µg/Liter)
MS (µg/Liter)
Ca S Fe Na Al Mg K Zn Pb Cu Ba P Y Mn B Sr Ti As Ni Sb Pd Cr Sn V Ce Co LI Rb Mo La Cd Nd Pr W
11909±112 3501±26 1135±13 556±8 523±13 463±7 442±6 399±4.5 244±4 215±0.9 106±2 92±0.6 55±1 36±1 29±0.55 29±0.2 9±0.3 5±0.5 4.9±0.2 3.8±0.1 3.8±0.2 3.6±1 2±0.06 2±0.05 1.4±0.03 1.±0.04 1.00±0.04 1±0.04 0.9±0.1 0.5±0.01 0.4±0.02 0.3±0.004 0.07±0.002 0.07±0.01
1.3±3.3 3235±52 17±0.3
12000 320 830 570 500 450 430 400 230 200 80 17 50 30
5±4 1.4±0.3 9. ±0.5 0.001±0.1 1.7±0.03 0.7±0.05 1.2±0.1 65±1.6 0.2±0.02 0.01+0.0125 26±0.5 0.04±0.01 0.3±0.13 4.3±0.25 0.15±0.2 2.5±0.014 0.24±0.005 0.24±0.03 0.12±0.01 0.03±0.01 0.04±0.004 0.004±0.005 0.02±0.01 0.09±0.005 0.2±0.015 0.008±0.001 0.004±0.002 0.007±0.0075 0.004±0.002 0.04±0.005
30
4.5 4 2 1.5 2.5
482 483 484 - 23 ACS Paragon Plus Environment
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485
Table 2. Comparison between Cation and redox active solutions for oxidative- and
486
inflammatory features
487
Cation solution
Redox Active
(% from MS)
(% from MS)
48.9
84
GSH
49
80
TNF-α
1
24
IL-6
1
37
ROS Tissue
47
73
Catalase
52
90
GPx
49
80
Cell count
488 489 490 491 492 493 494 495 496 497 498 499 500 501 502
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503
Corresponding Author: Yinon Rudich, Department of Earth and Planetary Sciences,
504
Weizmann Institute of Science, Rehovot, 76100, Israel. Phone: +972 8 934 4237; FAX +972-
505
8-934-4124; Mail:
[email protected].
506
NOTE. The authors declare no competing financial interest
507
ACKNOWLEDGEMENTS. This research was supported by the Estate of Nathan Minzly and
508
by the Grand Center at the Weizmann Institute. AR and J.JS collaboration was supported by
509
the Israel-US Binational Science Foundation (BSF).
510
Supporting information available. This information is available free of charge via the
511
internet at http://pubs.acs.org/
512
ABBREVIATIONS. PM, Particulate matter; ROS, Reactive oxygen species; ROFA,
513
Residual oil fly ash; BALF, broncho-alveolar lavage fluid; GSH, Glutathione; Nrf2, NF-E2-
514
related factor-2; NP, Nano-particle; IT, Intra-tracheal instillation; DCFDA, 2’,7’–
515
dichlorofluorescin diacetate; MDA, Malondialdehyde; MS, Metal solution; ARE, Antioxidant
516
response
517
Intraperitoneal
518
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