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Toxicology Letters 225 (2014) 12– 19

Contents lists available at ScienceDirect

Toxicology Letters

jou rn al hom ep age: www.elsev ier .com/ locate / tox le t

ytoplasmic p21CIP1/WAF1, ERK1/2 activation, and cytoskeletalemodeling are associated with the senescence-like phenotype afterirborne particulate matter (PM10) exposure in lung cells

esennia Sánchez-Péreza, Yolanda I. Chirinob, Álvaro Román Osornio-Vargasc,uis A. Herrerad, Rocío Morales-Bárcenasa, Alejandro López-Saavedraa,melda González-Ramíreza, Javier Mirandae, Claudia María García-Cuellara,∗

Instituto Nacional de Cancerología (INCan), Subdirección de Investigación Básica, San Fernando No. 22, Tlalpan, 14080 México, D.F., MexicoUnidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Los Reyes Iztacala, CP 54090 Tlalnepantla,stado de México, MexicoDepartment of Pediatrics, University of Alberta, 1048 RTF, 8308 114St Edmonton, Edmonton, AB T6G 2V2, CanadaUnidad de Investigación Biomédica en Cáncer, INCan; Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM),exico, Mexico

Instituto de Física, Universidad Nacional Autónoma de México, A.P. 20-364, 01000 Mexico, D.F., Mexico

i g h l i g h t s

Cells A549 exposed to PM10 from an industrial zone and a commercial zone with different composition.PM10 from both zones induced a senescence-like phenotype without cell cycle arrest.PM10 induced an increase in F-actin stress fibers.PM10 induced cytoplasmic p21CIP1/WAF1, ERK1/2 activation, and cytoskeleton remodeling.p21CIP1/WAF1 cytoplasmic retention may favor proliferation despite of senescence events.

r t i c l e i n f o

rticle history:eceived 14 May 2013eceived in revised form 22 October 2013ccepted 9 November 2013vailable online 26 November 2013

eywords:articulate matterM10

a b s t r a c t

The exposure to particulate matter with a mean aerodynamic diameter ≤10 �m (PM10) from urban zonesis considered to be a risk factor in the development of cancer. The aim of this work was to determine if PM10

exposure induces factors related to the acquisition of a neoplastic phenotype, such as cytoskeletal remod-eling, changes in the subcellular localization of p21CIP1/WAF1, an increase in �-galactosidase activity andchanges in cell cycle. To test our hypothesis, PM10 from an industrial zone (IZ) and a commercial zone (CZ)were collected, and human adenocarcinoma lung cell cultures (A549) were exposed to a sublethal PM10

concentration (10 �g/cm2) for 24 h and 48 h. The results showed that PM10 exposure induced an increasein F-actin stress fibers and caused the cytoplasmic stabilization of p21CIP1/WAF1 via phosphorylation at

145 146 202

ytoskeleton remodeling21CIP1/WAF1 cytoplasmicenescence-like

Thr and Ser and the phosphorylation of ERK1/2 on Thr . Changes in the cell cycle or apoptosiswere not observed, but an increase in �-galactosidase activity was detected. The PM10 from CZ causedmore dramatic effects in lung cells. We conclude that PM10 exposure induced cytoplasmic p21CIP1/WAF1

retention, ERK1/2 activation, cytoskeleton remodeling and the acquisition of a senescence-like phenotypetions

in lung cells. These altera

of PM10.

. Introduction

Air pollution from urban and industrial areas, specifically partic-late matter (PM), is hazardous to human health. Epidemiological

∗ Corresponding author. Tel.: +52 55 5628 0462; fax: +52 55 5628 0432.E-mail addresses: claudia.garciac@salud.gob.mx, garcue57@gmail.com

C.M. García-Cuellar).

378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.toxlet.2013.11.018

could have mechanistic implications regarding the carcinogenic potential

© 2013 Elsevier Ireland Ltd. All rights reserved.

evidence has identified associations between elevated levels of airpollution and a variety of health outcomes, including mortality(Pope et al., 1995), hospitalization, respiratory and cardiovasculardiseases, and the aggravation of asthma attacks (Brunekreef andForsberg, 2005). Evidence indicates that the source of PM with a

mean aerodynamic diameter ≤10 �m (PM10) determines the com-position of the PM, which is in turn related to the cellular effectsof exposure to PM. One of the clearest effects of PM10 exposureis the generation of reactive oxygen species, specifically hydroxyl

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adicals, which cause DNA damage. At least some of these effectsre related to high iron-content PM10 (García-Cuellar et al., 2002).n addition, PM10 containing high levels of endotoxin induces anncrease in cytokine production (e.g., IL-6) (Alfaro-Moreno et al.,002). Recently, attention has been focused on air pollution as aisk factor for lung cancer (Nafstad et al., 2003; Pope and Dockery,006), but the mechanisms involved in air pollution-related lungancer are not fully understood.

There is some evidence that PM10-induced genotoxicity coulde involved in the development of lung cancer. We previouslyemonstrated that sublethal PM10 concentrations have genotoxicffects on lung epithelial cells, evidenced by the induction ofNA double-strand breaks (DSBs), �H2A.X foci formation, and the

ecruitment of 53BP1. To protect the genome from PM10 exposure-nduced DNA damage, cells activated the ATM/ATR/Chk1/Chk2/p53athway, which promotes the adoption of a senescence-like stateSanchez-Perez et al., 2009). Senescent cells display remarkableharacteristics, such as an enhanced senescence-associated �-alactosidase activity, a cell cycle arrest in the G0–G1 phase,hanges in the cell cycle related to cytoskeletal remodelingAkakura et al., 2010), and changes in the inflammatory responseCalcabrini et al., 2004). Nevertheless, the association betweenM10-induced cytoskeletal remodeling and senescence remainsnknown. Additionally, some of the most significant alterations

n senescent cells include changes in the expression of tumor-uppressor genes, including p14Arf, p16INK4A, pRb, and p21CIP1/WAF1

Fang et al., 1999; Campisi, 2001; Zhang et al., 2007; Hoare andarita, 2013)

The p21CIP1/WAF1 protein is a member of the cyclin-dependentinase (CDK)-inhibitor (CKI) family, and its nuclear localizations linked to growth-inhibitory activity. The cytoplasmic stabiliza-ion of p21CIP1/WAF1 by phosphorylation at threonine 145 (Thr145)r serine 146 (Ser146) suppresses its growth-inhibitory activityWang et al., 2010; Zhu et al., 2010) and correlates with onco-enic transformation (Romanov et al., 2011), most likely becausehe phosphorylation of p21CIP1/WAF1 at Ser146 results in resistanceo apoptosis (Oh et al., 2007).

We are particularly interested in p21CIP1/WAF1 because it isssociated with cell senescence and tumor-promoting activityRoninson, 2002) and because its activity can be partially regulatedy p53 activation. We have previously reported that PM10 expo-ure induced p53 activation via phosphorylation at serine 15 (Ser15)Sanchez-Perez et al., 2009). The phosphorylation of p53 at Ser15

an be carried out by the extracellular signal-regulated kinases 1nd 2 (ERK1/2) (Persons et al., 2000). ERK1/2 are associated withtress fiber formation and are also important cellular proliferationarkers (Mebratu and Tesfaigzi, 2009). In addition, it is known

hat p21CIP1/WAF1 stabilization is associated with the activation ofhe ERK1/2 pathway (Arakawa et al., 2010). p21CIP1/WAF1 also hasunctions beyond regulating the cell cycle, including the remod-ling of the cytoskeleton through the increased polymerization oflamentous actin (F-actin) (Besson et al., 2004).

Based on the above information, we hypothesized that PM10ould induce a premature senescent-like state, the cytoplasmictabilization/retention of p21CIP1/WAF1 and activation of ERK1/2,nd cytoskeletal remodeling; all of these changes are related tohe acquisition of a neoplastic phenotype. We characterized thoseellular changes after exposure of lung epithelial cells to PM10.dditionally, to assess changes induced by PM samples with dif-

erent compositions, we compared PM10 from two different urbanones, one industrial zone and one commercial zone. This workhowed a relationship between PM10 exposure and F-actin stress

ber formation, cytoplasmic p21CIP1/WAF1 retention, and ERK1/2hr202 phosphorylation. These findings explain some of the pro-eoplastic cellular effects induced by PM10 and may explain howM10 participates in the carcinogenic processes.

y Letters 225 (2014) 12– 19 13

2. Materials and methods

2.1. PM10 sampling

PM10 was collected from two urban zones of Mexico City, oneindustrial zone (IZ) and one commercial zone (CZ) with a highlevel of traffic. The PM10 was collected using a high-volume par-ticle collector with a flux of 1.13 m3/min (GMW model 1 200 VFCHVPM10; Sierra Andersen, Smyrna, GA, USA). PM10 was recoveredon 3 �m pore size cellulose nitrate filters (Sartorius AG, Göettingen,Germany) for 3 days per week during the period of October 2004to May 2005. Particles were scraped off of the filters and storedas previously reported (Alfaro-Moreno et al., 2009). The particlesobtained during the collection were stored in endotoxin-free glassvials in a desiccator at 4 ◦C until use (Sanchez-Perez et al., 2009;Chirino et al., 2010).

2.2. Elemental analysis by particle-induced X-ray emission (PIXE)

Elemental analysis of the airborne particles was performed byPIXE using a 2.2 MeV proton beam produced by a 9SDH-2 Pel-letron accelerator (National Electrostatics Corp., Middleton, WI,USA) at the National Autonomous University (UNAM) Institute ofPhysics, as previously described (Miranda et al., 2000). The data areexpressed as the amount of metal detected in a pool of PM10 col-lected during one year (Bonner et al., 1998; Alfaro-Moreno et al.,2009).

2.3. Cell culture and exposure to PM10.

The A549 lung epithelial cells were obtained from the Amer-ican Type Culture Collection (ATCC, Rockville, MD, USA). Cellswere cultured in F12K medium (Invitrogen, Carlsbad, CA, USA)supplemented with 10% fetal bovine serum (FBS) in a 5% CO2atmosphere at 37 ◦C. All PM treatments were performed at 80%confluence in FBS-free medium using a sublethal concentrationof PM10 (10 �g/cm2). The IZ and CZ PM10 samples were unableto induce apoptosis or cytotoxicity at this concentration (Alfaro-Moreno et al., 2009; Sanchez-Perez et al., 2009) by 24 h or 48 h.After PM10 exposure, the cells were rinsed and collected for furtherevaluation (Sanchez-Perez et al., 2009; Chirino et al., 2010).

2.4. Cytotoxicity

The level of cytotoxicity was measured using the crystal violetassay (Kueng et al., 1989). Proliferating A549 cells were exposed to10 �g/cm2 PM10 for 24 h or 48 h. After exposure, the level of cyto-toxicity was determined by counting the residual number of cellswith crystal violet staining. The absorbance of the cells at 570 nmwas read using a plate reader (GENiosTM Plus, Tecan Trading AG,Switzerland). The data are expressed as the percentage of viablecells in three independent experiments.

2.5. Senescence-associated ˇ-galactosidase (SA-ˇ-gal) activity

Lung epithelial cells (5 × 103 cells) were seeded in 24-well cul-ture plates and exposed to PM10 (10 �g/cm2) for 24 h or 48 h. Theactivity of SA-�-gal after PM10 exposure was evaluated as describedin a previous report (Dimri et al., 1995). Briefly, the cells were fixedwith 4% paraformaldehyde at 37 ◦C for 1 h and then incubated with

staining solution [5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6 3H2O, pH 6.0;1 mg/mL of X-gal (Promega Madison WI, USA)] at 37 ◦C for 24 h. Thepercentage of SA-�-gal-positive cells was determined by counting100 cells per well under a microscope (Leica). The images were

1 icology Letters 225 (2014) 12– 19

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Table 1Elemental content of PM10 samples collected from two regions of Mexico City.

IZ CZ IZ CZ

Al 24.0 17 Cr 0.1 0.04Si 69.0 51.1 Mn 0.4 0.3P 1.3 0.5 Fe 12.3 10.1S 5.4 5.0 Ni 0.05 0.03Cl 1.3 0.05 Cu 0.2 0.3K 5.0 4.0 Zn 1.1 0.5Ca 25 15.0 Pb 0.22 0.2Ti 1.4 1.3

4 Y. Sánchez-Pérez et al. / Tox

aptured with a digital camera (Nikon), and the results are reporteds the percentage of the total number of cells scored.

.6. Cell cycle and apoptosis

After treatment, lung epithelial cells (2 × 106 cells per sample)ere collected and centrifuged (1000 × g for 5 min). The cells were

hen fixed with absolute ethanol at −20 ◦C. RNase A (20 �g DNase-ree) was added to the cells, and each sample was incubated at7 ◦C for 30 min. The cells were stained using 100 �L of propidium

odide. The cell cycle was analyzed by flow cytometry (FACSort,ecton Dickinson) using CellQuest and ModFit LT software.

The level of apoptosis was evaluated by staining cells withnnexin V-fluorescein isothiocyanate (Ex/Em = 495 nm/529 nm)nd propidium iodide. At each time point during PM10 expo-ure, cells were washed with PBS and centrifuged at 200 × g for

min. The pellet was suspended in 100 �L of annexin V solu-ion (Annexin V FLUOS Staining kit, Roche Diagnostics GmbH, cat.858777). Data acquisition and analysis were performed by flowytometry (FACSort, Becton Dickinson) using CellQuest software.

.7. Detection of F-actin and immunofluorescence staining of21CIP1/WAF1 and p21CIP1/WAF1 phosphorylated at Thr145 and Ser146

Cells were grown in 8-well culture plates (BD Falcon Franklinakes, NJ, USA) for the previously described treatments (Sec-ion 2.2). After the treatments, the cells were fixed in 4% (w/v)araformaldehyde, permeabilized with 0.2% Triton X-100 (Sigmahemicals Co., St. Louis, MO, USA) and then incubated with 0.1%odium borohydride (NaBH4) for 5 min to quench the autofluores-ence. Non-specific reactivity was blocked with 10% horse serum,% BSA, and 0.02% NaN3 in TBS. Cells exposed to 100 �M hydrogeneroxide (H2O2) were used as a positive control. To evaluate F-actintress fibers, the cells were incubated overnight with 500 ng/mLRITC-phalloidin (Sigma-Aldrich, St Louis, USA). The cover slipsere mounted on slides using ProLong Gold Antifade with DAPI

Molecular Probes). Images were captured using a microscope (Axiobserver Z1 DUO LSM 710 confocal system, Carl Zeiss).

The densitometry analysis was done on taken images of cellstaining with TRITC-phalloidin and were analyzed to optical densityOD) values using the Image J freeware program (rsb.info.nih.gov/ij)Girish and Vijayalakshmi, 2004) and compared to fluorescencebserved in control samples. Results were done in at least threendependent experiments and were expressed as folds of F-actin,elative to control.p21CIP1/WAF1 was evaluated in cells incubatedith a primary anti-human p21CIP1/WAF1 antibody (1:100) (Santaruz, CA, USA) in 1% BSA-TBS at 4 ◦C overnight. The secondary anti-ody used was a FITC-conjugated goat anti-mouse IgG (Jacksonmmuno Research, PA, USA).

For double labeling, a sequential immunofluorescence protocolas followed. For the first labeling step, the cells were incu-

ated with a 1:100 dilution of the primary anti-human p-p21Ser146) antibody (Santa Cruz, CA, USA); the secondary antibodyas a TRITC-conjugated donkey anti-goat IgG (Jackson Immunoesearch, PA, USA). For the second labeling step, the cells were incu-ated with a primary anti-human p-p21 (Thr145, Santa Cruz, CA,SA) and a secondary FITC-conjugated goat anti-rabbit IgG anti-ody (Jackson Immuno Research, PA. USA). The cover slips wereounted on slides using ProLong Gold Antifade with DAPI (Molec-

lar Probes). Images were captured using an inverted microscopeAxio Observer Z1 DUO LSM 710 confocal system, Carl Zeiss).

The densitometry analysis was done on taken images of cells

taining with rhodamine-labeled and fluorescein isothiocyanateFITC)—conjugated antibodies. The cytoplasm p21CIP1/WAF1 and21CIP1/WAF1, Ser146 and Thr145 retention was analyzed usinghe Image J freeware program (rsb.info.nih.gov/ij) (Girish and

Elemental content is expressed as micrograms per milligram of PM10 sample. IZ:PM10 collected from urban industrial zone; CZ: PM10 collected from urban commer-cial zone. Uncertainties range from 8% to 15%.

Vijayalakshmi, 2004). The results were represented as relative cyto-plasmic levels of p21CIP1/WAF1.

2.8. Western blot analysis of ERK1/2 and phosphorylated ERK1/2(Thr202)

After treatments, the cell cultures were rinsed twice with PBSand lysed with lysis buffer (20 mM Tris, 1% NP-40, and 150 mMNaCl, pH 8.0) containing a protease inhibitor cocktail (RocheMannheim, Germany). The protein content of each sample wasdetermined using the bicinchoninic acid protein assay (Smithet al., 1985), and the samples were maintained at −70 ◦C untiluse. The samples (containing 25 �g of protein) were mixed 1:1 inloading buffer (Bio-Rad, San Diego, CA, USA) containing 5% (v/v)�-mercaptoethanol. The proteins were separated using SDS-PAGEand transferred onto a nitrocellulose membrane. The membranewas blocked with 5% (w/v) dry milk in PBS containing 0.05% (v/v)Tween-20 (PBS-T) and then incubated with anti-ERK1/2 (Millipore)and anti-phosphorylated ERK1/2 (Thr202). The immunoreactivitywas visualized using an enhanced chemiluminescence detectionkit (Millipore). Actin was utilized as a loading control (kindly pro-vided by Dr. Manuel Hernández; Department of Cellular Biology,CINVESTAV-IPN, Mexico City, Mexico). We employed a 1:2000dilution of horseradish peroxidase-conjugated anti-mouse IgG(Amersham Pharmacia Biotech).

2.9. Statistical analysis

Data are expressed as the mean ± standard deviation (SD) of atleast three independent experiments. Different treatments werecompared using ANOVA followed by the Bonferroni test for indi-vidual comparisons of group means.

3. Results

3.1. Elemental content of PM10

The PIXE analysis revealed the presence of 16 elements in bothPM samples. Soil-associated elements (Al, Si, Ca, and Fe) were themost abundant components of PM10, followed by elements asso-ciated with anthropogenic sources (Cu, Zn, Pb, and Ni). The PM10samples from the IZ and the CZ differed in composition. The PM10from the IZ contained higher content of all elements except Cu thanthe PM10 from the CZ (Table 1).

3.2. PM10 from the IZ and the CZ induced an increase in thepercentage of SA-ˇ-gal-positive cells without causing cytotoxicity

or cell cycle arrest

Exposure to 10 �g/cm2 of PM10 from both zones did not inducecytotoxicity, as measured by violet crystal assay, after 24 h and 48 h

Y. Sánchez-Pérez et al. / Toxicology Letters 225 (2014) 12– 19 15

Table 2SA-�-gal positive and cytotoxicity in lung epithelial cells after PM10 exposure.

SA-�-gal positive cells (%) Cytotoxicity (% Viability)

24 h 48 h 24 h 48 h

CT 4 ± 1.7 1.7 ± 0.6 100 ± 0 100 ± 0IZ 8 ± 1.3*** 23 ± 3.2*** 98 ± 4 96 ± 4CZ 17 ± 2.4*** 43 ± 7*** 95 ± 3 96 ± 7

CT: control; IZ: PM10 collected from urban industrial zone; CZ: PM10 collectedfrom urban commercial zone. Lung epithelial cells exposed to PM10 (10 �g/cm2)during 24 h and 24 h after of exposure (48 h). Senescence-like phenotype was mea-sured by SA-�-gal activity. The cytotoxicity was evaluated by crystal violet. Data aremean ± SD of three independent experiments.

*** p < 0.001 vs. CT.

Table 3Cell cycle distribution and apoptosis after PM10 exposure in lung epithelial cells.

Cell cycle phase (%)

G0–G1 G2–M S % Apoptosis

24 hCT 57 ± 1.3 16 ± 1.0 11.1 ± 0.6 2 ± 1IZ 58.4 ± 3.2 14 ± 8 28 ± 10.0 3 ± 3.2CZ 58 ± 3.0 17 ± 5 25.3 ± 7.2 2.5 ± 2.4

48 h

CT 75.3 ± 3 16.2 ± 1 8.0 ± 3 1.70 ± 3IZ 71.1 ± 0.4 18.0 ± 2 11 ± 1.0 1.35 ± 0.3CZ 69 ± 1.0 18.0 ± 2 13.0 ± 2 1.4 ± 0.3

CT: control; IZ: PM10 collected from urban industrial zone; CZ: PM10 collected fromurban commercial zone. Lung epithelial cells were exposed to PM10 (10 �g/cm2)daa

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Fig. 1. PM10 exposure induced the remodeling of the cytoskeleton in A549 cells.Cell cultures were exposed to PM10 (10 �g/cm2) from the IZ and the CZ for 24 h and48 h. (A) F-actin stress fibers were stained with fluorescein-labeled phalloidin, andnuclei were visualized using DAPI. The images, which were acquired by fluorescencemicroscopy, show F-actin organization and are representative of three independentexperiments. (B) The quantitative results of actin fibers are expressed in arbitrary

uring 24 h and 24 h after of exposure (48 h). Cell cycle distribution was evalu-ted by flow cytometry and apoptosis was evaluated by Anexin V staining. Datare mean ± SD of three independent experiments.

Table 2). The exposure of lung epithelial cells to a sublethal concen-ration of IZ PM10 for 24 h induced a 2-fold increase in the numberf SA-�-gal-positive cells, whereas exposure to the PM10 from theZ induced a 4.5-fold increase in this senescence marker (Table 2).fter 48 h, the increases in the numbers of SA-�-gal-positive cellsere 13-fold and 26-fold for IZ and CZ PM10, respectively (Table 2).

ncreases in the levels of senescence markers are typically asso-iated with cell cycle alterations, but the cell cultures exposed toM10 from the IZ or the CZ for 24 or 48 h exhibited no changes in cellycle distribution (Table 3), level of viability (Table 2) or apoptosisTable 3).

.3. PM10 induced an increase in F-actin stress fibers

We clearly showed that exposure to PM10 from both zonesnduced an increase in the percentage of cells in a senescence-ike state. This phenotype is associated with increased levels of-actin stress fibers (Chen et al., 2000). Therefore, we determinedf the exposure to PM10 from the IZ or the CZ induces cytoskeletonemodeling using phalloidin. Interestingly, treatment with PM10rom both zones induced an increase significant of 2.8 (p < 0.01) and.7 (p < 0.001) folds, respectively, versus control of F-actin stressbers after 24 h (Fig. 1A and B). The cell cultures exposed to PM10

rom the CZ induced a major increase of 5.5 folds (p < 0.0001) of-actin stress fibers formation after 48 h (Fig. 1C and D).

.4. Cytoplasmic p21CIP1/WAF1 stabilization is induced by PM10rom the IZ and the CZ

Because actin is regulated by cytoplasmic p21CIP1/WAF1, we eval-CIP1/WAF1

ated the localization of p21 by immunofluorescence after

4 h and 48 h of PM10 exposure. The exposure to IZ PM10 induced slight significant stabilization (p < 0.01) of p21CIP1/WAF1 in theytoplasm of 2.3 folds. The CZ PM10 exposure induced a major

units of fluorescence. The data are reported as the means ± SDs of three independentexperiments. **p < 0.001 IZ vs. CT and ***p < 0001 CZ vs. CT to 24 h; ***p < 0.001 vs. CTto 48 h. ###p < 0.001 IZ vs. CZ to 48 h.

and significant stabilization of 3.1 folds (p < 0.001) of cytoplasmicp21CIP1/WAF1 after 24 h (Fig. 2A and B), becoming pronounced after48 h in cells exposed to PM10 from CZ (p < 0.001) the fluorescenceintensity increased 4.4 folds (Fig. 2C and B).

Given the p21CIP1/WAF1 stabilization in the cytoplasm after PM10exposure, we sought to determine if phosphorylation was relatedto this stabilization. The exposure of cells to PM10 from the IZand the CZ induced an increase of 1.6 (p < 0.01) and 2.4 (p < 0.001)folds, respectively, with respect to control of phosphorylation ofp21CIP1/WAF1 at Thr145 after 24 h (Fig. 3A and B). This effect wasstill observed after 48 h of exposure to PM10 from both zones:1.7 (p < 0.01) and 2.9 folds (p < 0.001), respectively, with respect tocontrol (Fig. 3C and D). The p21CIP1/WAF1 phosphorylated at Ser146

increased after exposure to PM10 from both zones (Fig. 3) andwere observed staining fluorescence in cytoplasm to 24 h and thisincrease were significant from both zones, IZ vs. Ct (3.5, p > 0.001)and CZ vs. CT (5.3, p < 0.001). The cytoplasmic p21CIP1/WAF1 phos-phorylated at Ser146 was sustained after 48 h and this cytoplasmicincrease was significant from both zones: IZ vs. Ct (2.9 folds,p < 0.001) and CZ vs. CT (4.6 folds, p < 0.001) (Fig. 3C and D).

3.5. PM10 induced proliferation related to ERK1/2phosphorylation

ERK1/2 up-regulates the expression of the p21CIP1/WAF1 protein(Arakawa et al., 2010), leading to its stabilization in the cytoplasm.We decided to investigate the phosphorylation state of ERK1/2. A

16 Y. Sánchez-Pérez et al. / Toxicology Letters 225 (2014) 12– 19

Fig. 2. Exposure to PM10 from the CZ induced p21CIP1/WAF1 stabilization in the cytoplasm. Cell cultures were exposed to PM10 (10 �g/cm2) from the IZ and the CZ for the24 h and 48 h. (A) The green color in the images represents p21CIP1/WAF1 cytoplasmic staining of cells exposed to PM10 from IZ and CZ to 24 h. (B) The quantitative resultswas expressed as relative p21CIP1/WAF1 level; *p < 0.001 IZ vs. CT and ***p < 0001 CZ vs. CT to 24 h;).(C) p21CIP1/WAF1 cytoplasmic staining of cells exposed to PM10 from IZ andC /WAF1 l* s are

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Z to 48 h. (D) The quantitative results of p21CIP1/WAF1 expressed as relative p21CIP1

p < 0.001 IZ vs. CT, ***p < 0001 CZ vs. CT and ###p < 0.001 IZ vs. CZ to 48 h. The imageeans ± SDs of three independent experiments.

ell culture exposed to IZ PM10 exhibited an increase in the level ofRK1/2 Thr202 phosphorylation after 24 h, and this effect persistedt 48 h (Fig. 4).

. Discussion

This study showed that exposure to PM10 from an IZ and aZ during 24 h increased the SA-�-gal activity in lung epithelialells, and this effect was still observed after 48 h of expo-ure. The cell culture exposed to PM10 from the CZ exhibited areater increase in the percentage of SA-�-gal-positive cells (4.5-old increase) than the cell culture exposed to PM10 from theZ, as shown in Table 2. Thus, our findings suggest that PM10rom both zones can induce a senescence-like state, most likelys a protective mechanism against DNA damage and oxidativetress.

We previously demonstrated that a sublethal concentration ofM10 (10 �g/cm2) can cause DNA double-strand breaks withouthanges in cell viability (Sanchez-Perez et al., 2009). We are par-icularly interested in the effects of non-lethal experimental PM10oncentrations because the epidemiological outcomes of PM10xposure are most likely the result of long term exposures to lowevels of PM10; for this reason, we selected a low PM10 concentra-ion to perform the experiments. It is also important to highlighthat the concentration used in this study could be comparable to

five day-exposure in humans (Pope et al., 2002; Li et al., 2003;ope and Dockery, 2006) and that we and others have reportedo alterations in cell viability when using 10, 20 and 40 �g/cm2 ofM10; therefore, 10 �g/cm2 is considered to be a sublethal PM10

evel. The data are reported as the means ± SDs of three independent experiments.representative of three independent experiments and the data are reported as the

concentration (Alfaro-Moreno et al., 2002; Sanchez-Perez et al.,2009; Chirino et al., 2010).

In this study, we did not observe changes in the cell cycle(i.e., cell cycle arrest) or the level of apoptosis in cells exposedto PM10 (Table 3). Previous reports have shown that exposure toPM10 at concentrations of 10 �g/cm2 (Sanchez-Perez et al., 2009),25 �g/cm2 or 50 �g/cm2 (Gualtieri et al., 2010) was unable toinduce apoptosis in lung epithelial cells. However, apoptotic effectscan be found using 160 �g/cm2 or higher concentrations (Alfaro-Moreno et al., 2002).

On the other hand, our results showed the ability of PM10to induce a senescence-like state, which can be a self-protectiveeffect in exposed cells but at the same time, it can be pro-tumorigenic stimulus. Even if this statement seems contradictory, ithas been demonstrated indeed, that senescence is an autonomoustumor suppressor mechanism, but this cellular event has also pro-tumorigenic effects on neighboring cells. This can be explainedsince senescent cells can release pro-inflammatory moleculesthat they act as autocrine pro-senescent stimuli but at the sametime, these pro-inflammatory signals can exert a paracrine pro-tumorigenic activity on neighboring cells (Campisi, 2001; Hoareand Narita, 2013).

To explore the potential mechanisms involved in increasedsenescence, we investigated the level of cytoskeletal remodelingbecause it has been demonstrated that morphological changes

related to senescence induce actin stress fibers (Chen et al., 2000).Exposure to PM10 was unable to modify the expression of actin(data no shown), but interestingly, we found a clear increase inthe number of F-actin stress fibers, which could be related to an

Y. Sánchez-Pérez et al. / Toxicology Letters 225 (2014) 12– 19 17

Fig. 3. Exposure to PM10 from the IZ and the CZ induced p21CIP1/WAF1 stabilization through the phosphorylation of p21CIP1/WAF1 at Ser146 and Thr145. Cell cultures were exposedto PM10 (10 �g/cm2) from the IZ and the CZ to 24 and 48 h. p21CIP1/WAF1 phosphorylated at Ser146 is labeled in red, and p21CIP1/WAF1 phosphorylated at Thr145 is labeled ingreen. Nuclei were visualized using DAPI. The merged images show the localization of phosphorylated p21CIP1/WAF1 in the cytosol. (A) Cells exposed to PM10 from IZ and CZand evaluated to p21CIP1/WAF1 Thr145 and p21CIP1/WAF1 Ser146 at 24 h. (B) The quantitative results of p21CIP1/WAF1 Thr145 and Ser146 were expressed as relative p-p21CIP1/WAF1

l 146 ** *** ### 4 h. Th 145 *** *** ##

C IP1/WA

t 48 h.

imcest2sg2

ift(ClWcrtWtsbascbp

evel; Ser p < 0.001 IZ vs. CT, p < 0001 CZ vs. CT and p < 0.001 IZ vs. CZ to 2ells exposed to PM10 from IZ and CZ and evaluated to p21CIP1/WAF1 Thr145 and p21C

o 24 h. Thr145 ***p < 0.0001 IZ vs. CT, ***p < 0001 CZ vs. CT and ##p < 0.001 IZ vs. CZ to

ncrease in the rate of actin polymerization. Changes in actin poly-erization are regulated by p21CIP1/WAF1, and this protein has a

ell-cycle inhibitory activity when localized to the nucleus. How-ver, other pathways could also contribute to the increase in actintress fibers, including the TGF-� pathway (Zhu et al., 2010) becausehis factor is secreted in response to PM exposure (Dagher et al.,005). Because the assembly of F-actin stress fibers requires theynergistic activation of the MAPK/Erk1/2 pathway and the smalluanosine triphosphatase Rho via its effector, Rock (Sabri et al.,004), the Rho/Rock pathway also could be involved.

Our findings indicate that p21CIP1/WAF1 was primarily locatedn the cytoplasm after PM10 exposure (Figs. 2 and 3). We alsoound that PM10 induced an increase in the cytoplasmic reten-ion of p21CIP1/WAF1 through phosphorylation at Thr145 and Ser146

Fig. 4). In addition, we observed that PM10 from the IZ and theZ induced slight fluorescence, suggesting a slight increases in the

evels of p21CIP1/WAF1 phosphorylated at Ser146 (Ben-Porath andeinberg, 2005) and that the impairment of the nuclear translo-

ation of p21CIP1/WAF1 prevents cell cycle arrest. Based on theseesults, we suggest that the inhibition of the nuclear transloca-ion of p21CIP1/WAF1 prevents its binding to pRb (Ben-Porath and

einberg, 2005), and the inhibition of this binding could be relatedo the lack of an effect on cell cycle arrest observed after PM10 expo-ure (Table 3). This cellular event could be harmful because it haseen reported that the cytoplasmic localization of p21CIP1/WAF1 isssociated with resistance to apoptotic stimuli (Oh et al., 2007). We

uggest that accumulation of p21CIP1/WAF1 protein could be asso-iated, at least in part, with the activation of the ERK pathwayy Raf/MEK/ERK (Coleman et al., 2003). Interestingly, cytoplasmic21CIP1/WAF1 localization is a marker of antineoplastic resistance

r p < 0.0001 IZ vs. CT, p < 0001 CZ vs. CT and p < 0.001 IZ vs. CZ to 24 h. (C)F1 Ser146 at 48 h. (D) *p < 0.01 IZ vs. CT, ***p < 0001 CZ vs. CT and ##p < 0.001 IZ vs. CZ

and is associated with poor prognosis in carcinomas (Abukhdeirand Park, 2008).

To identify a pathway that could explain the cytoplasmicp21CIP1/WAF1 retention, we investigated the effect of PM10 onERK1/2 activation. A clear increase in phosphorylation wasobserved (Fig. 4). We suggest that this effect could be mediatedby Akt activation because Akt induces the phosphorylation ofp21CIP1/WAF1 at the C-terminal Thr145/Ser146 residues, leading tocytoplasmic p21CIP1/WAF1 retention (Zhang et al., 2007). If Akt isinvolved in p21CIP1/WAF1 phosphorylation, it could be possible thatkappa B kinase � activates Akt (as has been previously reported),thus leading to cytoplasmic p21CIP1/WAF1 stabilization (Ping et al.,2006).

The observed effects in this study may be related to the differ-ences in composition between the PM10 from the CZ and the IZbecause it has been demonstrated that the composition of PM10is strongly related to its cytotoxicity. PM10 from the CZ is ableto induce stronger pro-inflammatory responses than PM10 fromthe IZ, as measured by the levels of TNF-�, IL-6, and E-selectin(Alfaro-Moreno et al., 2002). PM10 from the CZ has a higher con-tent of automotive emissions than the IZ (Villalobos-Pietrini et al.,1995), whereas PM10 from the IZ contains higher levels of transi-tion metals (Bonner et al., 1998). In this work, the content of metalsdid not explain the observed biological changes because the PM10with the higher levels of metals (IZ) did not have stronger effects.However, the high content of Cu in the PM10 from the CZ (Table 1)

could substantially contribute to the effects induced by these typesof PM10 (Kouassi et al., 2010; Huang et al., 2013). Additionally, ithas been previously demonstrated that metal content of PM10 isclearly associated with some biological effects. For example, PM10

18 Y. Sánchez-Pérez et al. / Toxicolog

Fig. 4. Exposure the PM10 from the IZ and the CZ induced the phosphorylation ofErk1/2. Cell cultures were exposed to PM10 (10 �g/cm2) from the IZ and the CZ for24 h, and then the PM10 was removed and replaced with normal growth mediumfor another 24 h. (A) Western blot showing non-phosphorylated and phosphorylatedERK1/2. This blot is representative of the blots from three independent experiments.(B) The quantitative results for the pERK1/2/ERK1/2 association are expressed inaiv

fILm(

pafiTttfoh

C

A

A

Qp1

R

A

A

rbitrary densitometric units. The data are reported as the means ± SDs of threendependent experiments. *p < 0.01 vs. CT to 24 h; #p < 0.01 vs. CT to 48 h, ##p < 0.01s. CT to 48 h.

rom CZ induced higher proinflammatory response than PM10 fromZ (Alfaro-Moreno et al., 2002; Rosas Perez et al., 2007; Manzano-eon et al., 2013) and the main metal content includes cooper,anganese, zinc, lead, potassium, nickel and sulfur, among others

Manzano-Leon et al., 2013).Finally, this study provides evidence that the source of PM10

lays an important role in the observed cellular effects, and were showing for the first time a relationship between F-actin stressber formation, cytoplasmic p21CIP1/WAF1 retention, and ERK1/2hr202 phosphorylation p21CIP1/WAF1, three events highly relatedo carcinogenic processes. These alterations could have mechanis-ic implications regarding the carcinogenic potential of PM10, andurther studies are needed to determine the detailed mechanismsf the effects of PM10 so that the potential adverse effects on humanealth can be elucidated.

onflicts of interest statement

The authors declare no conflicts of interest related to this work.ll authors have read and approved the manuscript.

cknowledgments

The authors express their gratitude to Yazmín Segura and Raúluintana, participants in the field campaign. This work was sup-orted by CONACyT-Mexico grants 43183-M, AC-2006-52830 and66727.

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