During the last week o June 2008, central and northern Caliornia experienced a major outbreak o wildires caused by a series o lightning strikes that was unprecedented in the past century in its extent and sever- ity, with transport o smoke over large dis- tances rom the res, especially in the Central Valley. A regional map with the location o the largest o these res illustrated is available rom the Caliornia Department o Forestry and Fire Protection (2008). Air quality in the region was severely aected by the smoke rom these res, and millions o people were exposed to quantities o wildire-generated particulate matter (PM) greatly in excess o the current PM standards. Hourly levels o PM with mass median aerodynamic diameter < 2.5 µm (PM2.5) at racy (near our sam- pling site) peaked at 160 µg/m3 (San Joaquin Valley Air Pollution Control District 2008), whereas hourly concentrations o PM with mass median aerodynamic diameter < 10 µm (PM10) peaked at 200 µg/m3. Further to the north in the Sacramento River Valley, closer to the major res, PM2.5 values o 262 µg/ m3 were reported on the same days. hus, PM with mass median aerodynamic diameter > 2.5 µm to < 10 µm (PM10‒2.5) and PM2.5 concentrations were greatly in excess o the Caliornia 24-hr average ambient air quality standards (PM10‒2.5, 50 µg/m3; PM2.5, 35 µg/ m3) and among the highest values reported at these stations since data have been collected or PM pollution in these size classications.

Tese observations raise concerns about the potential health impact o exposure to high levels o wildre PM, as the possible health eects associated with these acute exposures to PM rom wildres at these very high levels are not understood.

PM10‒2.5 and PM2.5 samples were obtained during the last week o June 2008, when the ires were at their worst, rom a U.S. Environmental Protection Agency (EPA) des- ignated National Air Emissions Monitoring Study site that was heavily impacted. he monitoring included data on PM10‒2.5 con- centrations logged every 2 min (Series FH 62C14 Beta Sampler; Termo Electron Corp., Franklin, MA). Peak value observed during the 2 days studied was 381 µg/m3, with val- ues between 200 and 380 µg/m3 logged rou- tinely over a period o several hours in the late aternoon and early evening o 26 June. Tus, the values reported at racy, the nearest San Joaquin Valley Air Pollution Control District monitoring site, probably underestimate the actual concentrations at our sampling site. Tis manuscript describes a toxicologic analysis o both the coarse and ine particles (PM10‒2.5 and PM2.5) collected during the 2-day period o peak air pollution during June 2008, and compares the toxicity o wildire PM with PM collected rom nearby ambient air under normal conditions during June 2007. his manuscript will demonstrate that the inherent toxicity on an equal-dose basis is greater or the wildre PM than that o PM rom normal

Particulate matter used in this study was col- lected with a high-volume air sampler (model GS2310; Andersen Instruments Inc., Smyrna, GA) equipped with a our-stage cascade impactor (series 230, Andersen Instruments Inc.) in the summer months rom a location in the northeast o the San Joaquin Valley in Caliornia. Slotted aluminum substrates (isch Environmental, Cleves, OH) were used or PM collection. Te nominal ow rate used or collection was 20 t3/min, with particle size cutos o 10.2, 4.2, 2.1, and 1.3 µm. For the purposes o this manuscript, we will reer to coarse PM as particles with a mass median aerodynamic diameter range o 10.2‒2.1 µm and ne PM as particles within 2.1‒1.3 µm. Ater collection, substrates rom each stage were weighed; particles were removed by scraping with a spatula and stored at –80°C in vials. Tirty minutes beore use, particles were suspended in phosphate-buered saline (PBS), pH 7.6 (Mediatech, Inc., Herndon, VA) at the desired concentration. Te nal pH o the resultant suspension was pH > 7.

Bioassay techniques in the mouse have been validated and optimized as described pre- viously (Wegesser and Last 2008). Briey, male BALB/c mice 8‒10 weeks o age (25‒30 g) were purchased rom Charles River Breeding Laboratories (Wilmington, MA). Mice were housed, our animals per cage, in ltered Bio- Clean acilities in the Animal Resources Center (University o Caliornia Davis, CA). Animals received water and standard eed (Purina Rat Chow) ad libitum and were allowed to accli- mate or 1 week beore any experi mental proce- dures. Te animals were kept on a 12-hr light/ dark cycle at room temperature (68‒70°F)

Address correspondence to J.A. Last, CCRBM, 6519 Genome and Basic Science Building, 451 Health Sciences Dr., Davis, CA 95616 USA. elephone: (530) 752-6230. Fax: (530) 752-8632. E-mail: jalast@ucdavis.edu

We thank F. Mitloehner, S. Cli, N. Kado, and D. Bennett or their assistance with access to the sampling site and PM sampling.

Tis study was unded in part by ES-07059 rom the National Institute o Environmental Health Sciences, U07/CCU906162 rom the National Institute or Occupational Saety and Health, and RD-83241401 rom the U.S. Environmental Protection Agency.

thousands o orest and brush res, giving rise to a week o severe re-related particulate air pollu- tion throughout the region. Caliornia experienced PM10‒2.5 (particulate matter with mass median aerodynamic diameter > 2.5 µm to < 10 µm; coarse ) and PM2.5 (particulate matter with mass median aerodynamic diameter < 2.5 µm; ne) concentrations greatly in excess o the air quality standards and among the highest values reported at these stations since data have been collected.

much more toxic to the lung on an equal weight basis than was PM collected rom normal ambient air in the region. oxicity was maniested as increased neutrophils and protein in lung lavage and by histologic indicators o increased cell infux and edema in the lung.

and 30‒70% relative humidity. All procedures were perormed under an Institutional Animal Care and Use Committee approved protocol. All animals used in this study were treated humanely and with regard or alleviation o suering.

Methods or intratracheal instillation o 50-µL suspensions o known amounts o PM into mice and evaluation o lung inam- mation are described in detail elsewhere (Wegesser and Last 2008). Bronchoalveolar lavage (BAL) and tissue were collected 24 hr ater PM instillation. Whole cell counts were perormed with whole lavage luid and a hemocytometer. Cells were separated rom supernatant by centriugation at 2,000 rpm in a benchtop centriuge and stained with Di-Quick (Fisher Scientiic; Kalamazoo, MI) or dierential cell counts. Protein con- tent o lavage luid supernatant was deter- mined by a colorimetric reaction with the Micro BCA Protein Assay Reagent Kit (Pierce Biotechnology, Rockord, IL). Lavaged lungs were xed at 30 cm pressure with 1% para- ormaldehyde in PBS or 1 hr or histopatho- logic assessment, ater staining with Harris’ hematoxylin and eosin, with an Olympus BH2 microscope connected to an OLY-750 Color Camera (Olympus; Center Valley, PA). Endotoxin in PM preparations was assayed by the Limulus amoebocyte lysate (LAL) assay (Wegesser and Last 2008).

San Diego, CA). All values are expressed as mean ± SE. Parametric analysis o data was conducted using analysis o variance with ukey’s post-test or multiple comparisons. Dierences were considered signicant i the

We ound no signicant dierences in total cells recovered by lung lavage between either untreated (data not shown) or saline-instilled controls and mice instilled with 10, 25, or 50 µg wildire PM10‒2.5 (Figure 1A). here was a signiicant increase in total lavageable cells with instillation o 100 µg PM10‒2.5 rom the wildre sample. In prior studies we have seen signicant dose-related increases (more than twice as many cells) in total lavageable cells rom mice instilled with 25 or 50 µg PM10‒2.5 rom ambient air samples collected rom this geographic area (Wegesser and Last 2008), so the lack o increase in total lavage- able cells seen between 10 and 50 µg PM10‒2.5 rom the wildre samples is unusual.

he cells lavaged rom lungs o control mice were 95‒100% macrophages, whereas lavage uid rom mice instilled 24 hr earlier with 50 µg PM10‒2.5 rom ambient air con- tained about 30% macrophages and 70% neutrophils (Wegesser and Last 2008). We ound 49 ± 15, 47 ± 18, and 57 ± 23% neutro- phils or mice instilled with 10, 25, or 100 µg

wildire PM10‒2.5, respectively (Figure 2A). hus, despite the lack o apparent increase in total cell numbers in the lung lavage rom the mice exposed to 10 or 25 µg PM10‒2.5 in Figure 1A, the mice responded to the wildre PM at the lowest and the highest doses tested. Te cell populations had shited to about hal neutrophils, which is not normal, despite total cell numbers remaining more or less constant. On an equal-dose basis, the wildire lavage samples contained signicantly lower numbers o macrophages than did lavage uid rom mice instilled with PM10‒2.5 collected rom normal ambient air (AA) during the same period 1 year earlier (Figure 2B; compare the responses to 25 and 50 µg wildire PM10‒2.5 with 25 AA and 50 AA, where 25 AA and 50 AA signiy the samples o 25 and 50 µg PM10‒2.5 rom normal ambient air). Direct LAL assay shows < 1 endotoxin unit (EU) o endotoxin/50 µg PM10‒2.5 preparation, ruling out a signicant role or lipopolysaccharide (LPS) in the genera- tion o the observed neutrophilic inammation, as Balb/C mice respond normally to endotoxin (Silvia and Urosevic 1999).

he lung inlammatory response to PM10‒2.5 rom the wildire diers rom the response to PM10‒2.5 rom ambient air. Because the total number o lavageable cells did not increase in the mice exposed to 10‒50 µg PM10‒2.5 (Figure 1A), and hal o the total cells were neutrophils, the wild- ire PM10‒2.5 must have caused a decrease in the numbers o macrophages in the lungs (Figure 1B). Note also that in the 100 µg wildre PM10‒2.5 sample, the total cells in the lavage were signiicantly increased and this increase was made up primarily o neutrophils (57% o the total cells). When compared with the 25 µg and 50 µg samples rom normal AA, all animals dosed with PM10‒2.5 rom the wildre (10, 25, 50, and 100 µg groups) had signicantly ewer macrophages in their lung lavage uid (Figure 2B). Tus, the most strik- ing aspect o the cell dierential counts in the mice exposed to the wildre-derived PM10‒2.5 is the relative absence o alveolar macrophages in their lungs, compared with PBS-instilled controls (Figure 2B). his accounts or the lower total cell count in these wildire PM-exposed lungs, suggesting that either the

11.0
8.0
6.0
4.0
2.000

6.0 5.0 4.0 3.0 2.0 1.00

amounts o PM10–2.5 or PM2.5 wildfre PM. All o the indicated values are signifcantly greater than PBS- instilled controls, which contained 0% polymorphonuclear leukocytes. (B) Number o macrophages in the lung lavage uid o mice instilled with either 25 or 50 µg PM: comparison o wildfre PM and normal AA PM collected 1 year earlier rom the same area. Values shown are mean ± SE.

100 75 50 250

2.5 2.0 1.5 1.0 0.50

1,400
1,200
1,000
800
600
400
2000

wildre PM10‒2.5 may be especially toxic to pulmonary alveolar macrophages or that these wildre PM10‒2.5 create a condition in which macrophages are difcult to extract rom the lungs by lavage, perhaps because o enhanced adherence to alveolar suraces.

As shown in Figure 3, there was signi- cantly more protein in the lavage uid super- natant rom mice instilled with 100 µg o the PM10‒2.5 raction, and a trend toward higher amounts o protein in all o the groups examined compared with controls. Te lack o a clear dose–response relationship in the data suggests that there is either a threshold in the observed response or that the lavage supernatant protein content is measuring a phenomenon more complex than simple uid transudation across the airway epithe- lial barrier in a damaged lung (Witschi and Last 2001). In contrast, we have not observed any signicant increase in lung lavage super- natant content o protein in mice exposed to PM10‒2.5 preparations rom normal AA col- lected rom the San Joaquin Valley.

We perormed similar experiments with PM2.5 preparations rom the wildre samples (Figures 1 and 3) collected simultaneously to acilitate direct comparisons o the two size ractions. We ound signiicantly more total cells in the mice instilled with 100 µg wildire PM2.5, with an apparent trend or dose response between 25 and 100 µg PM2.5 (Figure 1A). We observed signicantly ewer macrophages in the lung lavage luid rom mice instilled with either 50 or 100 µg wildre PM2.5 and comparable decreases o macro- phages in mice instilled with 100 µg wild- re PM10‒2.5 or PM2.5 (Figure 1B). Both the 50 µg and 100 µg samples caused signicant increases in the concentration o lung lavage supernatant protein in mice exposed to wild- ire PM2.5 preparations (Figure 3), with an apparent dose-related dierence in response to the 25 µg dose versus the 50- and 100-µg doses o PM2.5 tested. Te increase in amount o protein in the lung lavage supernatant was not signiicantly dierent between the mice instilled with 100 µg PM10‒2.5 or PM2.5.

As shown in Figure 4, a marked inux o cells composed o monocytes and neutrophils was observed in mice instilled with 100 µg wildre PM10‒2.5 within the peribronchial tis- sues o the airways, along with an increased cel- lularity o septal tissues in the lung parenchyma with notable accumulation o inlammatory cells in the centriacinar airspaces o the lungs. Occasional extravasation o red blood cells, along with patchy edema uid, was also noted in the alveolar airspaces. Increased lung tissue damage was noted with increasing doses o instilled particles or both PM2.5 and PM10‒2.5 (Figures 4‒7). In addition, wildre PM10‒2.5 particles induced greater histologic changes to the lungs or both the airways and the alveoli when compared with PM10‒2.5 particles col- lected under normal ambient conditions.

here exists extensive literature on epidemi- ologic studies and a much smaller literature on whole-animal studies o the heath eects o exposure to woodsmoke rom stoves, agri- cultural burning, wildres, and other sources (Naeher et al. 2007; Zeliko et al. 2002). Dubick et al. (2002) presented evidence o oxi- dative stress (lipid peroxidation) in lungs o rats acutely exposed (16 min) to whole woodsmoke by inhalation. Li et al. (1997) intratracheally instilled PM10‒2.5 collected rom AA in Scotland into rats and observed neutrophilic

inammation, increase in protein content, and oxidant stress (less glutathione) in lung lavage uid rom these animals, similar to our ndings in this study. Many authors have examined the toxicity and proinlammatory activity o PM10‒2.5 and/or PM2.5 by examination o their eects on cultured cells in vitro (e.g., Jiménez et al. 2002; Monn and Becker 1999; Veranth et al. 2004). Others have examined the toxicity o ractionated PM components to cultured cells (e.g., Adamson et al. 1999; Carter et al. 1997; Imrich et al. 2000). Specic toxicologic studies with PM isolated rom wildre smoke (e.g., Leonard et al. 2000; Jalava et al. 2006) are rare in the literature, presumably because o difculty in collecting such PM ractions.

he lungs o mice exposed to wildire PM10‒2.5 or PM2.5 in the present study showed signicant damage, as measured by histologic evaluation o inammatory cell inux or by relative neutrophil content or total protein content o lung lavage uid, compared with mice exposed to 10-old higher doses o nor- mal AA PM rom the same area. Te relative toxicity o PM10‒2.5 and PM2.5 seemed similar in these experiments, but we should note that use o the intratracheal instillation route would mask dierences in actual PM dosage to the lung o these dierent size ractions when they were inhaled. Based on the responses o mice to the 10 µg dose o wildre PM10‒2.5 or PM2.5 compared with the response to