Single Exposure to near Roadway Particulate Matter Leads to

Subscriber access provided by University of Otago Library

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Environmental Science & Technology

1

Single exposure to near roadway particulate matter leads to confined

2

inflammatory and defense responses: possible role of metals

3 4

Michal Pardo,1 Martin M. Shafer,2 Assaf Rudich,3 James J. Schauer,2 and Yinon Rudich1*

5 6 7 8

1

9

76100, Israel. E-mail: [email protected]

Department of Earth and Planetary Sciences, Weizmann Institute of Science, Rehovot

10

2

11

Madison, WI, USA

12

3

13

National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-

14

Sheva, Israel

Environmental Chemistry and Technology Program, University of Wisconsin-Madison,

Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, and the

15 16 17 18 19

Key words: Roadway metals, Air Pollution, Oxidative Stress, Nrf2, Inflammation, Intra-

20

tracheal installation

21

-1ACS Paragon Plus Environment


22

ABSTRACT

23

Inhalation of traffic-associated atmospheric particulate matter (PM2.5) is recognized as a

24

significant health risk. In this study we focused on a single ("sub-clinical response") exposure

25

to water-soluble extracts from PM collected at a roadside site in a major European city, to

26

elucidate potential components that drive pulmonary inflammatory, oxidative and defense

27

mechanisms, and their systemic impacts. Intra-tracheal instillation (IT) of the aqueous

28

extracts induced a 24h inflammatory response characterized by increased broncho-alveolar

29

lavage fluid (BALF) cells and cytokines (IL-6 and TNF-α), increased reactive oxygen species

30

production, but insignificant lipids and proteins oxidation adducts in mice' lungs. This local

31

response was largely self-resolved by 48h, suggesting that it could represent a sub-clinical

32

response to everyday-level exposure. Removal of soluble metals by chelation markedly

33

diminished the pulmonary PM-mediated response. An artificial metal solution (MS)

34

recapitulated the PM extract response. The self-resolving nature of the response is associated

35

with activating defense mechanisms (increased levels of catalase and glutathione peroxidase

36

expression), observed with both PM extract and MS. In conclusion, metals present in PM

37

collected near roadways are largely responsible for the observed transient local pulmonary

38

inflammation and oxidative stress. Simultaneous activation of the antioxidant defense

39

response may protect against oxidative damage.

40 41 42 43 44 45 46 -2ACS Paragon Plus Environment

Page 2 of 30

Page 3 of 30

47


TOC

48 49



50

INTRODUCTION:

51

Outdoor air pollution by fine atmospheric particles is a significant public health risk

52

that contributes to over 3.2 million premature deaths annually worldwide, thus placing

53

outdoor air pollution among the top 10 global mortality risks.1 The effect of near roadway air

54

pollution on human health is especially significant in major cities, with respiratory and

55

cardiovascular diseases being mostly implicated in response to continued/repeated exposure

56

to air pollution.2-7 In an effort to limit roadway-related air pollution, the common regulations

57

have focused on reducing tailpipe emissions. Among the various particulate matter (PM)

58

constituents,8 some metals9, 10 are potentially cytotoxic and can contribute to organ and tissue

59

damage and injury.3, 11,

60

sources of these metals it is expected that their roadway emissions are largely associated with

61

re-suspended road dust that contains brake wear, tire wear, and crustal elements.13-16

62

Recently, Verma et al. showed that all major components of fine PM, including metals,

63

contribute to reactive oxygen species (ROS) generation, and that exposure to aerosol

64

components can catalyze production of oxidants in vivo.17 Consistently, PM0.25-2.5 samples

65

collected in Beirut near a freeway contained high concentrations of metals and trace elements

66

(including Mn, Cr, Cu, and Ba) that correlated with in vitro ROS-activity in macrophage

67

cells.18 Several toxicological studies established associations between various coal and

68

residual oil fly ash (ROFA; a transition metal-dominated sample) metals and the PM-induced

69

inflammation in lung tissue and broncho-alveolar lavage fluid (BALF).19-21 Metals such as

70

Pb, Cr, V, Fe, Cu, Mn, Sr and Ba, were measured at relatively high levels near roadway

71

samples.15,

72

presents a major challenge in identifying specific components that are most biologically

73

active and/or leading to pathogenesis of disease. Identifying these components is essential for

74

appropriate mitigation policies that could reduce their public health impact.

22, 23, 24

12

Yet, since tailpipe emissions from mobile sources are not major

The heterogeneity of chemical species and different roadway sources


Page 4 of 30

Page 5 of 30


75

Oxidative damage is caused when the production of ROS exceeds their removal by

76

antioxidant defense mechanisms, and different cellular constituents are consequently

77

oxidized. Transition metals present in PM can induce ROS production, and the ensuing

78

oxidative stress was proposed to constitute a major mechanism mediating PM -induced health

79

impacts.6 Recent studies reported that PM-derived water-soluble transition metals (e.g. Fe,

80

Ni, Cu, Cr, Mn, Zn and V) significantly correlate with the oxidative potential of airborne PM

81

across different urban areas and particle size ranges.25,

82

oxidative stress by up-regulating a distinct array of cyto-protective genes responsible for the

83

cells’ antioxidant and detoxification capacity.27 Some of these genes act to maintain cellular

84

reduced glutathione (GSH) levels and high reduced/oxidized glutathione (GSH/GSSG)

85

content. A master regulator of this anti-oxidant response is the transcription factor NF-E2-

86

related factor-2 (Nrf2).27 Indeed, several studies demonstrated that Nrf2 is activated in vivo

87

and in vitro after exposure to diesel exhaust particles, PM, or to engineered nano-particles

88

(NP).28, 29 Moreover, the degree to which Nrf2-dependent antioxidant response functionally

89

meets the oxidative stress challenge posed by PM/NPs likely determines cell fate.29

26

Cells or tissues can respond to

90

In this study we aimed at identifying the in vivo role PM-derived transition metals

91

from collected roadside PM in mediating pulmonary inflammatory and oxidative stress

92

response, as well as the Nrf2-related antioxidant/detoxification defense mechanism. Metals

93

present in the water-soluble extracts from collected roadway particles were either chelated

94

from the extracts, or in complementary experiments, a water metal solution containing the

95

same composition and concentrations of the PM-derived metals was used, and the effect on

96

pulmonary and systemic inflammation and oxidant stress and defense were assessed. We

97

used a single, low-dose exposure by intra-tracheal instillation (IT) to elicit a transient

98

inflammatory/oxidative response rather than a toxic/full-blown disease-promoting exposure



99

level, to model pulmonary response to common levels of daily exposure to roadway PM in an

100

urban environment.

101

MATERIALS AND METHODS

102

PM collection and water extract preparation. Samples were collected from a roadside

103

monitoring site in central London (Marylebone Road near Baker Street) in early July 2012. A

104

Hi-Vol sampler (Tisch (TE-230) Hi-Vol Environmental Impactor Sampler), configured for

105

three particle size cuts (10 µm) and fitted with pre-cleaned mixed-

106

cellulose ester (MCE) substrates, was deployed to obtain two time-segregated samples.

107

Samplers were run continuously for 3-4 days at nominal flow rates of 1.2 m3 min-1, sampling

108

5000 m3 of air, and collecting from 50-300 mg of PM onto the MCE filter media.

109

Sub-sections of the MCE filter-collected PM3 were digested for total metals

110

(microwave aided mixed-acid digestion) and extracted with high-purity Milli-Q (18 mΩ)

111

water using an initial sonication period of 15 minutes (min) followed by 16h of continuous

112

agitation at room temperature in dark conditions and then finally another 15 min sonication.

113

At the end of the extraction period, aliquots of the suspension were removed and distributed

114

for various assays and analyses (PM extract), and the remaining suspension volume

115

centrifuged (6600 rpm for approximately 1 min) and then filtered through 0.22 µm

116

polypropylene syringe filters. Soon after filtration, an aliquot of the filtered sample was

117

processed through a miniature column of carefully pre-cleaned Chelex 100 resin (Bio-Rad

118

#143-2832, imino-diacetate in sodium form) to remove/isolate Chelex-labile (truly soluble

119

ions and “loosely” bound) metals (Chlx). The Chelex treatments were performed with 0.2g of

120

Chelex loaded into 1.5 mL polypropylene SPE reservoirs at a sample flow rate of 1 mL/min.

121

Method blanks replicated the processing procedure (Control). The extracts and digests of the

122

PM were subjected to a broad range of characterization tools, including: total and water-

123

soluble elements [ICPMS (SF-ICPMS)]; soluble ions (K+, Na+, NH4+, SO42-, NO3-, Cl-) by -6ACS Paragon Plus Environment

Page 6 of 30

Page 7 of 30


124

IC; soluble organic carbon; and several toxicity assays. The TOTAL elemental mass

125

extracted from the London PM (microwave-aided acid digestion), was normalized to the PM

126

mass to enable a direct comparison with similarly normalized data from the unfiltered water

127

extract data. Using a chemical species mass model, the total mass that these elements

128

contribute to the total PM was calculated. The filters show more than 80% recovery of the

129

target metals and inorganic species.

130

Metal solution (MS) preparation. To investigate the influence of metals on the bioassays, a

131

series of pure metal solutions were prepared to mimic the concentration of metals in the

132

extracts of the PM samples. The specific metal mixtures were: (a) major cations/anions to

133

ensure that we captured the bulk metals and ion-strength [Ca, Mg, Na, K, Ba, Sr, S, P], (b)

134

major and minor cationic/anionic redox-active transition metals [Fe, Mn, Cu, V, Ni, Cr], (c)

135

major non-redox active metals [Zn, Pb, Al], and (d) several minor elements of significance in

136

roadway emissions [Y, Ce, Pt and Pd]. The metal mixture solutions as defined in (a)–(d) were

137

prepared from high-purity individual element NIST-traceable stock standards (High Purity

138

Standards, Charleston SC). Metal concentrations in the 4 solutions were such that after a 10-

139

fold dilution of the solution the individual metal concentrations in the exposure solution

140

would be at the target levels. We used the cations solution (a) and the redox active metals (b)

141

MSs were prepared in acid-washed LDPE bottles using high-purity (18.2 MΩ) water–

142

sufficient acidity remained from the original stock solutions to maintain metal stability. The

143

exposure matrix provided adequate buffering to sustain optimum bioassay pH. See Table 1

144

for concentrations in the metals solution.

145

Mice treatments. Male C57BL/6 (7-8 weeks) mice were housed under standard light/dark

146

conditions and were given access to food and water ad libitum. Experiments were approved

147

by the Animal Care and Use Committee of the Weizmann institute of Science. Mice were

148

randomly divided into groups (minimum, n=6), where different water extracts and solutions



149

were used: water extract from urban London PM sampling (“PM extract”); water extract from

150

urban London PM sampling after chelation of soluble metals (“Chlx”) (non-specific sorption

151

of hydrophobic organic carbon to the resin backbone may occur, however the loss is minimal

152

30

153

the London water extract (“MS”). Pure metal solutions containing major cations: (Ca, Mg,

154

Na, K, Ba, Sr, S and P) (“Cations”) and redox active metals (V, Ni, Cu, Fe, Mn, and Cr)

155

(“Redox Active”). Control animals were exposed to blank extracts suspension in a

156

corresponding manner, another control group exposed mice to the PM extract using

157

intraperitoneal injection (i.p) (Supporting Information (SI), Table S1.). Prior to the mice

158

exposure, salts glucose medium, consisting of 500mM HEPES, 1MNaCl, 50mM KCl, 20mM

159

CaCl2 and 50mM dextrose PH 7.2, were added to sub-samples of the PM extracts, then pH

160

was 7.5. After i.p anaesthesia with ketamine/xylazin (10mg/kg and 5mg/kg body weight,

161

respectively), each mouse was placed on an inclined plastic platform; the dosing of

162

particulate suspension was performed while the tongue was pulled out with forceps to prevent

163

the mouse from swallowing the sample. The extract dose was delivered onto the vocal cords

164

and afterwards, the nostrils were covered, forcing the mouse to inspire the instilled particle

165

suspension. Each mouse received a single dose of 50µl PM water extract corresponding to

166

10µg PM2.5. Twenty-four hours (h) or 48h after instillation, all mice recovered the procedure

167

with no apparent health consequences. Then, mice were anesthetized again by i.p

168

ketamine/xylazin (20mg/kg and 10mg/kg body weight, respectively); blood was taken from

169

the retro-orbital vein for serum-cytokine analysis. Whole-body perfusion with PBS was

170

performed, the lungs and tracheas were exposed by dissection, tracheal cannula was inserted.

171

Lungs were lavaged with PBS solution twice. Cells were separated from the BALF by

172

centrifugation (500 × g, 5 min). The supernatant was removed and the cells were re-

173

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


Page 8 of 30

Page 9 of 30


174

Turk’s solution (crystal violet and 6% acetic acid) exclusion method.

175

Tissue preparation. Immediately after taking the BALF, the left lung was removed and

176

divided to several sections which were snap-frozen in liquid nitrogen for future analysis. The

177

remaining lung tissue (50-70mg) was rinsed with PBS and placed in a buffer containing

178

0.25M sucrose, 1mM EDTA, and 5mM HEPES, pH 7.4, followed by mincing the tissue

179

using scissors and pestle. The resulting homogenate was centrifuge 14,000g for 5 min to

180

remove cell debris.31 The supernatant was immediately analyzed for ROS and subsequent

181

protein determination using the Bradford method.32

182

ROS analysis. ROS were detected using 2’,7’–dichlorofluorescin diacetate (DCFDA)

183

detection kit (Abcam, UK) on fresh BAL cells and fresh lung tissue according to manufacture

184

instructions. Briefly, fresh cells and tissue homogenates were incubated with DCFDA for 30

185

min at 37 °C, fluorescence was measured with maximum excitation and emission spectra of

186

495 and 529 nm respectively in a microplate reader. Hydrogen peroxide (H2O2) and Tert-

187

Butyl hydrogen peroxide (TBHP) were used as positive controls. Calibration curves were

188

performed with H2O2 on lung Tissue and BAL cells respectively (SI, Figure S1A).

189

Immunoassay Measurements for Cytokines Secretion. Mouse ELISA kits (eBioscience,

190

Affymetrix, CA) were used according to the manufacture’s instructions to measure IL-6, and

191

TNF-α secretion in BALF and in the serum. Concentrations of IL-6, and TNF-α were

192

calculated from a calibration curve with known (supplied) standards. The absorbance of the

193

samples was measured at 450 and 570 nm by a microplate reader.

194

Lipid peroxidation. The extent of lipid oxidation in lung tissue was evaluated using the

195

thiobarbituric acid (TBA) method,33 producing a pink pigment. The lung homogenate was

196

mixed 1:1 with 50% trichloroacetic acid to precipitate the protein. After centrifugation, the

197

supernatant was reacted with TBA (280mg in 100 ml) in a boiling water bath for 10min.



198

After cooling, the absorbance at 532 nm using was measure using a spectrophotometer. The

199

values obtained were compared with a series of MDA tetrabutylammonium salt standard

200

curve (Sigma-Aldrich, St. Louis, MO, USA) (SI, Figure S1B).

201

Protein carbonyl content. Protein carbonyl content, a general indicator for protein

202

oxidation, in lung tissue homogenates was measured using protein carbonyl calorimetric

203

assay (Cayman chemicals, Ann Arbor, Michigan, USA) according to manufacture

204

instructions. Briefly, the reaction between 2,4, dinitrophenyl hydrazine (DNPH) and protein

205

carbonyl forming a Schiff base and produces a corresponding hydrazone was measured. The

206

amount of protein-hydrazone was quantified at 370 nm using a spectrophotometer. Protein

207

carbonyl content was calculated using the extinction coefficient of 22,000 M− 1 cm− 1 and

208

expressed in nmol/mg protein.

209

Measurements of total GSH. Total glutathione (oxidized + reduced; GSSG + GSH) in the

210

lung tissues was determined using a glutathione assay kit (Sigma-Aldrich). Briefly, snap

211

frozen lung tissues were minced and suspended in 5% 5-sulfosalicylic acid and incubated in a

212

reaction mixture containing 95mM potassium phosphate buffer, 0.95mM EDTA, 48µM

213

NADPH, 0.031mg/ml DTNB, 0.115units/ml glutathione reductase, and 0.24% 5-

214

sulfosalicylic acid. The absorbance was measured at 412 nm using a microplate reader with

215

kinetic read. The standard curve was obtained from absorbance of the diluted commercial

216

GSH.

217

Gene expression; RNA isolation, reverse transcription (RT), and real-time PCR. Total

218

RNA was extracted from lung tissue, using RNeasy RNA kit (QIAGENE, Hilden, Germany).

219

RT and real-time PCR were performed for quantification of mRNA expression using the

220

SYBR Green PCR mix (Applied Biosystems, Foster City, CA, USA) in StepOnePlus RT

221

PCR instrument. Total RNA (0.5 µg) was reverse-transcribed to cDNA using random

222

hexamers. The following cycling conditions were used in the PCR: 40 cycles at 95 °C for - 10 ACS Paragon Plus Environment

Page 10 of 30

Page 11 of 30


223

30 s and 60 °C for 30 s and extension at 72 °C for 30 s. β-actin and 18S mRNA were used as

224

endogenous controls. Melt-curve analysis showed a single peak for the primer sets of genes,

225

indicating no primer dimer formation. RNA samples were used as non-RT controls to exclude

226

interference by genomic DNA contamination. Slopes were – 3.4 for both target and

227

endogenous control genes. cDNA dilutions for the samples were set such that ∆CT between

228

the target and the endogenous control genes was no more than five cycles. Primers were

229

purchased from Sigma-Aldrich. (Rehovot, Israel) and are described in the following table (SI,

230

Table S2).

231

Statistical analysis. Results are expressed as means ± standard errors of the mean (SEM).

232

One-way ANOVA was used in multivariable analyses. Statistical evaluation was performed

233

with OriginLab (Data Analysis and Graphing Software, Northampton, MA, USA).

234

Differences were considered significant at probability level p < 0.05 using the Fisher

235

protected least-significant difference method.

236 237

RESULTS AND DISCUSSION

238

In this study we used 4 solutions: 1. “control”: Method blanks that replicated the entire

239

procedure, without PM2.5 extracts; “PM extract”: Water extracts from PM collected in a

240

near roadway station in London. 3. “Chlx”: Chelated PM extracts, where the water extracts

241

passed through a Chelex column to remove soluble metals. 4. “MS”: an artificial metal

242

solution with similar metal concentrations to those present in the “PM extract” solution.

243

Detailed description of the preparation is given in the Methods section above.

244

Inflammatory responses in BALF

245

Cell count in BALF was used to estimate the pulmonary inflammatory response to a

246

single, low-dose, intra-tracheal instillation of extracts of water-soluble material from - 11 ACS Paragon Plus Environment


247

collected near-roadway PM (“PM extract”). After a single low dose exposure of mice to

248

urban PM water extracts, the total cell number in the BALF increased significantly after 24h,

249

consistent with previous studies.24,

250

specific systemic inflammatory response, as the same dose of PM extract administered intra-

251

peritoneally (i.p) failed to elicit a similar response (SI, Table S1). To test the contribution of

252

metals to this biological response to PM extract, two complementary experiments were

253

performed: i. Metals were removed from the PM-water extract by filtration and chelation on

254

a column (“Chlx” solution), and the response was compared to that of the “PM extract”

255

solution. ii. An artificial aqueous metal solution (“MS” solution) containing only the metals,

256

with the same content and concentration as the PM extract itself (Table 1) was prepared to

257

test if it could recapitulate the response to the “PM extract” solution. The Chlx column indeed

258

greatly depleted the PM water extract from metals by >95% (Table 1). Exposure to the metal-

259

depleted solution induced a markedly lower increase in BALF cell content 24h following IT

260

administration (Figure 1). Conversely, the MS solution induced a significantly increased

261

BALF cellular content that was not statistically different from the response after exposure to

262

the PM extract (Figure 1).

34, 35

This response in the BALF did not reflect a non-

263

To determine if the 24h time point represents the initiation of a fulminant pulmonary

264

inflammatory response, BALF were obtained also 48h after extract administration. While

265

after 24h BALF cell counts were >3-fold higher in response to PM extracts compared to

266

control solution, at 48h only a ~50% increase in cell number was observed (results not

267

shown), suggesting significant and near-complete resolution of this inflammatory response of

268

the lungs by 48h post instillation.

269

To better characterize the inflammatory response observed in response to PM extract

270

and MS exposure, and to assess its systemic impact, TNF-α and IL-6 levels were measured in

271

the BALF and in the serum. Paralleling the BALF cell content, both cytokines were elevated - 12 ACS Paragon Plus Environment

Page 12 of 30

Page 13 of 30


272

several fold in BALF in response to either PM water extract or to the artificial MS solution

273

compared to control (Figure 2A, B). Moreover, removal of metals by chelation fully

274

abrogated this pro-inflammatory response to the PM extract. This inflammatory response was

275

fully resolved by 48h post installation (data not shown). The onset of pulmonary

276

inflammation evident here by the increased cytokines levels and cell numbers in the BALF is

277

consistent with a previous study,36 and with previous observations of increased neutrophil

278

numbers in BALF after exposure to road tunnel dust,37 urban air5 and mineral dust.3

279

Furthermore, our results strongly implicate water-soluble metals in this response, and indeed,

280

metals were suggested as major contributors to the inflammatory response to PM2.5

281

samples.3, 35

282

It has been suggested that cytokines produced in response to PM inhalation in the lung

283

may enter the blood stream and activate inflammatory response in remote organs (e.g heart,

284

adipose tissue, and muscle).38,

285

evaluated to assess the systemic impact of this low-level PM extract exposure. Serum TNF-α

286

and IL-6 levels remained unaltered in the 24h and 48h post-exposure to the PM water extracts

287

(SI, Figure S2).

39

Therefore, levels of TNF-α and IL-6 in serum were also

288

Overall, the results described so far suggest that inhalation of PM water extract at the

289

dose used induce a transient, self-resolving inflammatory response without gross systemic

290

inflammatory impact. Consistently, BALF cell number and cytokines levels showed

291

approximately the same magnitude of increase (about 3.5 fold increase for the cell count and

292

IL-6, and 2.5 for TNF-α), implying that increased cytokines content can mainly be attributed

293

to the higher cell recruitment into the lungs, with immune activation playing a minor role.

294

This further supports the notion that the exposure level used reflects a low-dose, transient,

295

local reaction in the lungs that minimally manifests systemically.

296

Oxidative stress alternation in the lung tissue - 13 ACS Paragon Plus Environment


297

ROS levels were measured in fresh tissue homogenates using 2’,7’–dichlorofluorescin

298

diacetate (DCFH-DA) probe where H2O2 and Tert-Butyl Hydrogen Peroxide (TBHP) were

299

used as positive controls. A major possible limitation of this assay is that the probe can be

300

oxidized by a number of free radicals and is not ROS-specific. DCFH competes with

301

antioxidants as well as other cell macromolecules and therefore serves only as an estimate of

302

ROS generation. Nevertheless, in this experiment we show that 24h following exposure to the

303

PM water extract and to the artificial MS induced ROS generation, while the control and

304

Chlx samples did not (Figure 3A). After 48h, ROS levels were lower than after 24h exposure

305

and not significantly higher than those of the control (results not shown). These observations

306

are in agreement with studies showing that PM-associated metals can increase oxidative

307

stress and inflammation, thereby contributing to adverse health effects of air pollution.6, 9, 31,

308

36, 40

309

H2O2 in a surrogate lung fluid.41

Moreover, transition metals such as Cu and quinones from PM produced high levels of

310

When ROS production exceeds the capacity to neutralize them, oxidatively-modified

311

diverse cellular macromolecules, including nucleic acids, proteins and lipids accumulate,

312

indicating oxidative damage.42 To assess whether the inflammatory and oxidant effect of PM

313

extract and MS resulted in measurable oxidant damage, lipid peroxidation and protein

314

carbonyl products were measured. Neither the PM water extract, nor the artificial MS,

315

changed lung tissue malondealdehyde (MDA) levels, 24 and 48h after installation (Figure 3B,

316

the 48h results are not shown). Similarly, the protein carbonyl content in the lung tissue did

317

not change following exposure to PM extracts (Figure 3C). The lipid peroxidation finding

318

differs from the prominent increase in peroxidative damage observed following exposure of

319

mice' lungs to low doses (15 µg) urban PM collected in Buenos Aires,5 or exposure of rat

320

lungs to coal dust PM10,43 and in RAW 264.7 cells exposed to welding fumes (which

321

contains a complex mix of metal oxide particles).44 Collectively, these data indicate that - 14 ACS Paragon Plus Environment

Page 14 of 30

Page 15 of 30


322

exposure to PM containing high concentrations of metals induces lung lipids damage. Our

323

data suggests that the levels of metals in the soluble fraction of the roadside PM we used

324

(source: London) were too low (10µg dose) to induce demonstrable pulmonary lipid

325

peroxidation toxicity response upon exposure to a single dose, despite mounting a measurable

326

inflammatory and ROS production response. This suggests that PM extract or MS at the

327

conditions used did not exceed the antioxidant/detoxifying capacity of the tissue.

328

Nrf2 phase II antioxidant defense

329

We next explored whether the apparent compensation against tissue damage indeed

330

engaged activation of defense mechanisms. To this end, we assessed phase II detoxifying and

331

antioxidant enzymes - classical targets of Nrf2, the principal regulator of this major cellular

332

antioxidation mechanism, acting by binding the antioxidant response element (ARE) in its

333

target genes' promoters.45 Indeed, PM water extract and MS produced a significant (2.5-fold)

334

rise in the stress-induced protein heme oxygenase-1 (HO-1) (Figure 4A). Antioxidant genes

335

such as catalase and glutathione peroxidase (GPx) also increased by 2.5 fold (each,

336

respectively) (Figure 4B, and C, respectively) with exposure to PM extract and MS at 24h. In

337

contrast, after removal of the metals by chelation (Chlx), the levels of these antioxidant genes

338

were similar to the control (Figure 4A, B, C). An additional, major cellular antioxidant

339

regulated by Nrf2 is the tripeptide GSH. We observed that the PM water extract and the MS

340

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


347

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

Page 16 of 30

Page 17 of 30


372

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


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


Page 18 of 30

Page 19 of 30


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



433

Page 20 of 30

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


Page 21 of 30


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



Page 22 of 30

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


Page 23 of 30


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


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


Page 24 of 30

Page 25 of 30


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

REFRENCES

519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535

1. Lim, S. S.; Vos, T.; Flaxman, A. D.; Danaei, G.; Shibuya, K.; Adair-Rohani, H.; Amann, M.; Anderson, H. R.; Andrews, K. G.; Aryee, M.; Atkinson, C.; Bacchus, L. J.; Bahalim, A. N.; Balakrishnan, K.; Balmes, J.; Barker-Collo, S.; Baxter, A.; Bell, M. L.; Blore, J. D.; Blyth, F.; Bonner, C.; Borges, G.; Bourne, R.; Boussinesq, M.; Brauer, M.; Brooks, P.; Bruce, N. G.; Brunekreef, B.; Bryan-Hancock, C.; Bucello, C.; Buchbinder, R.; Bull, F.; Burnett, R. T.; Byers, T. E.; Calabria, B.; Carapetis, J.; Carnahan, E.; Chafe, Z.; Charlson, F.; Chen, H.; Chen, J. S.; Cheng, A. T.; Child, J. C.; Cohen, A.; Colson, K. E.; Cowie, B. C.; Darby, S.; Darling, S.; Davis, A.; Degenhardt, L.; Dentener, F.; Des Jarlais, D. C.; Devries, K.; Dherani, M.; Ding, E. L.; Dorsey, E. R.; Driscoll, T.; Edmond, K.; Ali, S. E.; Engell, R. E.; Erwin, P. J.; Fahimi, S.; Falder, G.; Farzadfar, F.; Ferrari, A.; Finucane, M. M.; Flaxman, S.; Fowkes, F. G.; Freedman, G.; Freeman, M. K.; Gakidou, E.; Ghosh, S.; Giovannucci, E.; Gmel, G.; Graham, K.; Grainger, R.; Grant, B.; Gunnell, D.; Gutierrez, H. R.; Hall, W.; Hoek, H. W.; Hogan, A.; Hosgood, H. D., 3rd; Hoy, D.; Hu, H.; Hubbell, B. J.; Hutchings, S. J.; Ibeanusi, S. E.; Jacklyn, G. L.; Jasrasaria, R.; Jonas, J. B.; Kan, H.; Kanis, J. A.; Kassebaum, N.; Kawakami, N.; Khang, Y. H.; Khatibzadeh, S.; Khoo, J. P.; Kok, C.; Laden, F.; Lalloo, R.; Lan, Q.; Lathlean, T.; Leasher, J. L.; Leigh, J.; Li, Y.; Lin, J. K.; Lipshultz, S. E.; London, S.; Lozano, R.; Lu, Y.; Mak, J.; Malekzadeh, R.; Mallinger, L.;

element;

HO-1,

Hemeoxygenase-1;

GPx,

Glutathione


peroxidase;

i.p,


536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584

Marcenes, W.; March, L.; Marks, R.; Martin, R.; McGale, P.; McGrath, J.; Mehta, S.; Mensah, G. A.; Merriman, T. R.; Micha, R.; Michaud, C.; Mishra, V.; Mohd Hanafiah, K.; Mokdad, A. A.; Morawska, L.; Mozaffarian, D.; Murphy, T.; Naghavi, M.; Neal, B.; Nelson, P. K.; Nolla, J. M.; Norman, R.; Olives, C.; Omer, S. B.; Orchard, J.; Osborne, R.; Ostro, B.; Page, A.; Pandey, K. D.; Parry, C. D.; Passmore, E.; Patra, J.; Pearce, N.; Pelizzari, P. M.; Petzold, M.; Phillips, M. R.; Pope, D.; Pope, C. A., 3rd; Powles, J.; Rao, M.; Razavi, H.; Rehfuess, E. A.; Rehm, J. T.; Ritz, B.; Rivara, F. P.; Roberts, T.; Robinson, C.; RodriguezPortales, J. A.; Romieu, I.; Room, R.; Rosenfeld, L. C.; Roy, A.; Rushton, L.; Salomon, J. A.; Sampson, U.; Sanchez-Riera, L.; Sanman, E.; Sapkota, A.; Seedat, S.; Shi, P.; Shield, K.; Shivakoti, R.; Singh, G. M.; Sleet, D. A.; Smith, E.; Smith, K. R.; Stapelberg, N. J.; Steenland, K.; Stockl, H.; Stovner, L. J.; Straif, K.; Straney, L.; Thurston, G. D.; Tran, J. H.; Van Dingenen, R.; van Donkelaar, A.; Veerman, J. L.; Vijayakumar, L.; Weintraub, R.; Weissman, M. M.; White, R. A.; Whiteford, H.; Wiersma, S. T.; Wilkinson, J. D.; Williams, H. C.; Williams, W.; Wilson, N.; Woolf, A. D.; Yip, P.; Zielinski, J. M.; Lopez, A. D.; Murray, C. J.; Ezzati, M.; AlMazroa, M. A.; Memish, Z. A., A comparative risk assessment of burden of disease and injury attributable to 67 risk factors and risk factor clusters in 21 regions, 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 2012, 380, (9859), 2224-60. 2. Adamkiewicz, L.; Badyda, A. J.; Gayer, A.; Mucha, D., Disability-adjusted life years in the assessment of health effects of traffic-related air pollution. Adv Exp Med Biol 2015, 834, 15-20. 3. Happo, M. S.; Salonen, R. O.; Halinen, A. I.; Jalava, P. I.; Pennanen, A. S.; Dormans, J. A.; Gerlofs-Nijland, M. E.; Cassee, F. R.; Kosma, V. M.; Sillanpaa, M.; Hillamo, R.; Hirvonen, M. R., Inflammation and tissue damage in mouse lung by single and repeated dosing of urban air coarse and fine particles collected from six European cities. Inhal Toxicol 2010, 22, (5), 402-16. 4. Liu, E.; Yan, T.; Birch, G.; Zhu, Y., Pollution and health risk of potentially toxic metals in urban road dust in Nanjing, a mega-city of China. The Science of the total environment 2014, 476-477, 522-31. 5. Martin, S.; Fernandez-Alanis, E.; Delfosse, V.; Evelson, P.; Yakisich, J. S.; Saldiva, P. H.; Tasat, D. R., Low doses of urban air particles from Buenos Aires promote oxidative stress and apoptosis in mice lungs. Inhal Toxicol 2010, 22, (13), 1064-71. 6. Yang, W.; Omaye, S. T., Air pollutants, oxidative stress and human health. Mutat Res 2009, 674, (1-2), 45-54. 7. Zhu, Y.; Kuhn, T.; Mayo, P.; Hinds, W. C., Comparison of daytime and nighttime concentration profiles and size distributions of ultrafine particles near a major highway. Environmental science & technology 2006, 40, (8), 2531-6. 8. Murphy, G., Jr.; Rouse, R. L.; Polk, W. W.; Henk, W. G.; Barker, S. A.; Boudreaux, M. J.; Floyd, Z. E.; Penn, A. L., Combustion-derived hydrocarbons localize to lipid droplets in respiratory cells. Am J Respir Cell Mol Biol 2008, 38, (5), 532-40. 9. Ghio, A. J., Biological effects of Utah Valley ambient air particles in humans: a review. J Aerosol Med 2004, 17, (2), 157-64. 10. Rondini, E. A.; Walters, D. M.; Bauer, A. K., Vanadium pentoxide induces pulmonary inflammation and tumor promotion in a strain-dependent manner. Part Fibre Toxicol 2010, 7, 9. 11. Ekstrand-Hammarstrom, B.; Magnusson, R.; Osterlund, C.; Andersson, B. M.; Bucht, A.; Wingfors, H., Oxidative stress and cytokine expression in respiratory epithelial cells exposed to well-characterized aerosols from Kabul, Afghanistan. Toxicol In Vitro 2013, 27, (2), 825-33.


Page 26 of 30

Page 27 of 30

585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634


12. Peltier, R. E.; Cromar, K. R.; Ma, Y.; Fan, Z. H.; Lippmann, M., Spatial and seasonal distribution of aerosol chemical components in New York City: (2) road dust and other tracers of traffic-generated air pollution. J Expo Sci Environ Epidemiol 2011, 21, (5), 484-94. 13. Amato, F.; Cassee, F. R.; Denier van der Gon, H. A.; Gehrig, R.; Gustafsson, M.; Hafner, W.; Harrison, R. M.; Jozwicka, M.; Kelly, F. J.; Moreno, T.; Prevot, A. S.; Schaap, M.; Sunyer, J.; Querol, X., Urban air quality: the challenge of traffic non-exhaust emissions. Journal of hazardous materials 2014, 275, 31-6. 14. Harrison, R. M.; Jones, A. M.; Gietl, J.; Yin, J.; Green, D. C., Estimation of the contributions of brake dust, tire wear, and resuspension to nonexhaust traffic particles derived from atmospheric measurements. Environmental science & technology 2012, 46, (12), 65239. 15. Schauer, J. J.; Lough, G. C.; Shafer, M. M.; Christensen, W. F.; Arndt, M. F.; DeMinter, J. T.; Park, J. S., Characterization of metals emitted from motor vehicles. Res Rep Health Eff Inst 2006, (133), 1-76; discussion 77-88. 16. van der Gon, H. A.; Gerlofs-Nijland, M. E.; Gehrig, R.; Gustafsson, M.; Janssen, N.; Harrison, R. M.; Hulskotte, J.; Johansson, C.; Jozwicka, M.; Keuken, M.; Krijgsheld, K.; Ntziachristos, L.; Riediker, M.; Cassee, F. R., The policy relevance of wear emissions from road transport, now and in the future--an international workshop report and consensus statement. Journal of the Air & Waste Management Association 2013, 63, (2), 136-49. 17. Verma, V.; Fang, T.; Guo, H.; King, L.; Bates, J. T.; Peltier, R. E.; Edgerton, E.; Russell, A. J.; Weber, R. J., Reactive oxygen species associated with water-soluble PM2.5 in the southeastern United States: spatiotemporal trends and source apportionment. Atmos. Chem. Phys. Discuss. 2014, 14, (13), 19625-19672. 18. Daher, N.; Saliba, N. A.; Shihadeh, A. L.; Jaafar, M.; Baalbaki, R.; Shafer, M. M.; Schauer, J. J.; Sioutas, C., Oxidative potential and chemical speciation of size-resolved particulate matter (PM) at near-freeway and urban background sites in the greater Beirut area. The Science of the total environment 2014, 470-471, 417-26. 19. Kodavanti, U. P.; Schladweiler, M. C.; Richards, J. R.; Costa, D. L., Acute lung injury from intratracheal exposure to fugitive residual oil fly ash and its constituent metals in normo- and spontaneously hypertensive rats. Inhal Toxicol 2001, 13, (1), 37-54. 20. Hutchison, G. R.; Brown, D. M.; Hibbs, L. R.; Heal, M. R.; Donaldson, K.; Maynard, R. L.; Monaghan, M.; Nicholl, A.; Stone, V., The effect of refurbishing a UK steel plant on PM10 metal composition and ability to induce inflammation. Respir Res 2005, 6, 43. 21. Arantes-Costa, F. M.; Lopes, F. D.; Toledo, A. C.; Magliarelli-Filho, P. A.; Moriya, H. T.; Carvalho-Oliveira, R.; Mauad, T.; Saldiva, P. H.; Martins, M. A., Effects of residual oil fly ash (ROFA) in mice with chronic allergic pulmonary inflammation. Toxicol Pathol 2008, 36, (5), 680-6. 22. Riley, M. R.; Boesewetter, D. E.; Kim, A. M.; Sirvent, F. P., Effects of metals Cu, Fe, Ni, V, and Zn on rat lung epithelial cells. Toxicology 2003, 190, (3), 171-84. 23. Zhao, J.; Lewinski, N.; Riediker, M., Physico-Chemical Characterization and Oxidative Reactivity Evaluation of Aged Brake Wear Particles. Aerosol Science and Technology 2014, 49, 65-74. 24. Schauer, J. J.; Majestic, B. J.; Sheesley, R. J.; Shafer, M. M.; Deminter, J. T.; Mieritz, M.; Committee, H. E. I. H. R., Improved source apportionment and speciation of low-volume particulate matter samples. Res Rep Health Eff Inst 2010, (153), 3-75; discussion 77-89. 25. Saffari, A.; Daher, N.; Shafer, M. M.; Schauer, J. J.; Sioutas, C., Global perspective on the oxidative potential of airborne particulate matter: a synthesis of research findings. Environmental science & technology 2014, 48, (13), 7576-83. 26. Shafer, M. M.; Perkins, D. A.; Antkiewicz, D. S.; Stone, E. A.; Quraishi, T. A.; Schauer, J. J., Reactive oxygen species activity and chemical speciation of size-fractionated - 27 ACS Paragon Plus Environment


635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683

atmospheric particulate matter from Lahore, Pakistan: an important role for transition metals. J Environ Monit 2010, 12, (3), 704-15. 27. Itoh, K.; Chiba, T.; Takahashi, S.; Ishii, T.; Igarashi, K.; Katoh, Y.; Oyake, T.; Hayashi, N.; Satoh, K.; Hatayama, I.; Yamamoto, M.; Nabeshima, Y., An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun 1997, 236, (2), 313-22. 28. Calderon-Garciduenas, L.; Franco-Lira, M.; Mora-Tiscareno, A.; Medina-Cortina, H.; Torres-Jardon, R.; Kavanaugh, M., Early Alzheimer's and Parkinson's disease pathology in urban children: Friend versus Foe responses--it is time to face the evidence. Biomed Res Int 2013, 2013, 161687. 29. Pardo, M.; Shuster-Meiseles, T.; Levin-Zaidman, S.; Rudich, A.; Rudich, Y., Low cytotoxicity of inorganic nanotubes and fullerene-like nanostructures in human bronchial epithelial cells: relation to inflammatory gene induction and antioxidant response. Environmental science & technology 2014, 48, (6), 3457-66. 30. Bowles, K. C.; Apte, S. C.; Batley, G. E.; Hales, L. T.; Rogers, N. J., A rapid Chelex column method for the determination of metal speciation in natural waters. Analytica chimica acta 2006, 558, (1-2), 237-245. 31. Best, T. M.; Fiebig, R.; Corr, D. T.; Brickson, S.; Ji, L., Free radical activity, antioxidant enzyme, and glutathione changes with muscle stretch injury in rabbits. J Appl Physiol (1985) 1999, 87, (1), 74-82. 32. Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72, 248-54. 33. Ohkawa, H.; Ohishi, N.; Yagi, K., Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979, 95, (2), 351-8. 34. Antonini, J. M.; Zeidler-Erdely, P. C.; Young, S. H.; Roberts, J. R.; Erdely, A., Systemic immune cell response in rats after pulmonary exposure to manganese-containing particles collected from welding aerosols. J Immunotoxicol 2012, 9, (2), 184-92. 35. Wallenborn, J. G.; Schladweiler, M. J.; Richards, J. H.; Kodavanti, U. P., Differential pulmonary and cardiac effects of pulmonary exposure to a panel of particulate matterassociated metals. Toxicol Appl Pharmacol 2009, 241, (1), 71-80. 36. Deng, X.; Rui, W.; Zhang, F.; Ding, W., PM2.5 induces Nrf2-mediated defense mechanisms against oxidative stress by activating PIK3/AKT signaling pathway in human lung alveolar epithelial A549 cells. Cell Biol Toxicol 2013, 29, (3), 143-57. 37. Gerlofs-Nijland, M. E.; Boere, A. J.; Leseman, D. L.; Dormans, J. A.; Sandstrom, T.; Salonen, R. O.; van Bree, L.; Cassee, F. R., Effects of particulate matter on the pulmonary and vascular system: time course in spontaneously hypertensive rats. Part Fibre Toxicol 2005, 2, (1), 2. 38. Simkhovich, B. Z.; Kleinman, M. T.; Kloner, R. A., Air pollution and cardiovascular injury epidemiology, toxicology, and mechanisms. J Am Coll Cardiol 2008, 52, (9), 719-26. 39. Mancuso, P., Obesity and lung inflammation. J Appl Physiol (1985) 2010, 108, (3), 722-8. 40. Snow, S. J.; De Vizcaya-Ruiz, A.; Osornio-Vargas, A.; Thomas, R. F.; Schladweiler, M. C.; McGee, J.; Kodavanti, U. P., The effect of composition, size, and solubility on acute pulmonary injury in rats following exposure to Mexico city ambient particulate matter samples. J Toxicol Environ Health A 2014, 77, (19), 1164-82. 41. Charrier, J. G.; McFall, A. S.; Richards-Henderson, N. K.; Anastasio, C., Hydrogen peroxide formation in a surrogate lung fluid by transition metals and quinones present in particulate matter. Environmental science & technology 2014, 48, (12), 7010-7.


Page 28 of 30

Page 29 of 30

684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718


42. McCaskill, M. L.; Kharbanda, K. K.; Tuma, D. J.; Reynolds, J. D.; DeVasure, J. M.; Sisson, J. H.; Wyatt, T. A., Hybrid malondialdehyde and acetaldehyde protein adducts form in the lungs of mice exposed to alcohol and cigarette smoke. Alcohol Clin Exp Res 2011, 35, (6), 1106-13. 43. Kania, N.; Mayangsari, E.; Setiawan, B.; Nugrahenny, D.; Tony, F.; Wahyuni, E. S.; Widodo, M. A., The Effects of Eucheuma cottonii on Signaling Pathway Inducing Mucin Synthesis in Rat Lungs Chronically Exposed to Particulate Matter 10 (PM10) Coal Dust. J Toxicol 2013, 2013, 528146. 44. Leonard, S. S.; Chen, B. T.; Stone, S. G.; Schwegler-Berry, D.; Kenyon, A. J.; Frazer, D.; Antonini, J. M., Comparison of stainless and mild steel welding fumes in generation of reactive oxygen species. Part Fibre Toxicol 2010, 7, 32. 45. Kaspar, J. W.; Niture, S. K.; Jaiswal, A. K., Nrf2:INrf2 (Keap1) signaling in oxidative stress. Free Radic Biol Med 2009, 47, (9), 1304-9. 46. Sun, Q.; Tan, D.; Zhou, Q.; Liu, X.; Cheng, Z.; Liu, G.; Zhu, M.; Sang, X.; Gui, S.; Cheng, J.; Hu, R.; Tang, M.; Hong, F., Oxidative damage of lung and its protective mechanism in mice caused by long-term exposure to titanium dioxide nanoparticles. J Biomed Mater Res A 2012, 100, (10), 2554-62. 47. Messier, E. M.; Day, B. J.; Bahmed, K.; Kleeberger, S. R.; Tuder, R. M.; Bowler, R. P.; Chu, H. W.; Mason, R. J.; Kosmider, B., N-acetylcysteine protects murine alveolar type II cells from cigarette smoke injury in a nuclear erythroid 2-related factor-2-independent manner. Am J Respir Cell Mol Biol 2013, 48, (5), 559-67. 48. Liu, X.; Meng, Z., Effects of airborne fine particulate matter on antioxidant capacity and lipid peroxidation in multiple organs of rats. Inhal Toxicol 2005, 17, (9), 467-73. 49. Rice, T. M.; Clarke, R. W.; Godleski, J. J.; Al-Mutairi, E.; Jiang, N. F.; Hauser, R.; Paulauskis, J. D., Differential ability of transition metals to induce pulmonary inflammation. Toxicol Appl Pharmacol 2001, 177, (1), 46-53. 50. Melamed, S.; Lalush, C.; Elad, T.; Yagur-Kroll, S.; Belkin, S.; Pedahzur, R., A bacterial reporter panel for the detection and classification of antibiotic substances. Microb Biotechnol 2012, 5, (4), 536-48. 51. Charrier, J. G.; Anastasio, C., On dithiothreitol (DTT) as a measure of oxidative potential for ambient particles: evidence for the importance of soluble transition metals. Atmos Chem Phys 2012, 12, (5), 11317-11350. 52. Ntziachristos, L.; Froines, J. R.; Cho, A. K.; Sioutas, C., Relationship between redox activity and chemical speciation of size-fractionated particulate matter. Part Fibre Toxicol 2007, 4, 5.

719


Environmental Science & Technology Page 30 of 30 PM extract containing metals

Inﬂammation

ROS

Antioxidant Oxidative response/ ACS Paragon Plus stressEnvironment phase II damage genes

Single Exposure to near Roadway Particulate Matter Leads to

Recommend Documents