A total of 24 BALB/C mice (12 male and 12 female, 6–8 weeks old, 16–18 g; Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were exposed to EG (6 males and 6 females) or clean air (6 males and 6 females). Mice were housed at 25°C with 50–80% relative humidity under a 12:12-h light/dark cycle, with access to food and water ad libitum. The mice were placed in a cage in a sealed chamber (Fig. 1). Gas exposure (GE) was performed for 5 h/day (between 9.00 a.m. and 2.00 p.m.), 5 consecutive days per week, for 6 weeks in an exposure chamber (Fig. 1A). Following 30 days of treatment, mice were sacrificed, and the lung tissues were collected for further molecular experiments (Fig. 1B and C). EG was collected from a gasoline internal combustion engine and mixed with fresh air in a ratio of 1:10 using the electromechanical injection system. Subsequently, the mixed gas was injected into the chamber at a flow rate of 3 l/min. All animal experiments were approved by The Animal Care and Use and Ethics Committee of Jilin University (Changchun, China).

A male BALB/C mouse, housed under clean air conditions (no GE), was sacrificed, and the fresh lung tissues were cut into ~1 mm³ pieces (Fig. 1E and F). Subsequently, the lung tissues were seeded in a 6-well plate and dried in a flow tissue culture hood for 15 min until the tissues attached to the plate surface. A total of 500 µl Dulbecco's modified Eagle's medium (HyClone; GE Healthcare Life Sciences, Logan, MT, USA) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences) and antibiotics (100 mg/ml streptomycin and 100 U/ml penicillin; HyClone; GE Healthcare Life Sciences) was added to the culture plates and the plates were incubated at 37°C in a humidified incubator with 5% CO₂ (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The medium was replaced every other day until mLFC proliferation was observed under a light microscope (ECLIPSE Ts2; Nikon Corporation, Tokyo, Japan).

mLFCs (5×10³) were treated with 3 µM BaA, BaP or BEP (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 72 h, as previously described (20). Subsequently, cell proliferation was determined using the water-soluble tetrazolium salt (WST-1) cell proliferation reagent (Beyotime Institute of Biotechnology, Haimen, China). Briefly, 20 µl WST-1 reagent was added to 200 µl cell culture medium and incubated at 37°C in the dark for 2.5 h. The optical density (OD) at 450 and 630 nm was measured using a microplate reader (Synergy H1; BioTek Instruments, Inc., Winooski, VT, USA). Final OD values were calculated using the following formula: OD_final=OD₄₅₀-OD₆₃₀-OD_blank.

Following treatment with 3 µM BaA, BaP or BEP for 72 h, cell apoptosis was determined by staining mLFCs with propidium iodide (PI) and Annexin V-fluorescein isothiocyanate (FITC). Cells (1×10⁶) were washed with PBS and centrifuged at 200 × g for 5 min at room temperature. The cellular pellet was suspended in 50 µl Annexin V solution containing 5 µl Annexin V-FITC and 5 µl PI for 15 min at room temperature, provided in the Annexin V-FITC Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Data acquisition and data analysis were performed using a flow cytometer (FACScan; Becton-Dickinson and Company, Franklin Lakes, NJ, USA) with the FlowJo FACS analysis software (version 10.0; FlowJo LLC, Ashland, OR, USA).

Total protein was extracted from mLFCs treated with BaA and BaP using a lysis buffer containing 50 mM Tris/acetate (pH 7.4), 1 mM EDTA, 0.5% Triton X-100, 150 mM sodium chloride and 0.1 mM phenylmethane sulfonyl fluoride. Total protein was quantified using a Bradford protein assay kit (Beyotime Institute of Biotechnology). Proteins (20 µg/lane) were separated by 8–10% SDS-PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The PVDF membranes were incubated in Tris-buffered saline buffer containing 0.5% Tween-20 (Sigma-Aldrich; Merck KGaA) and 5% skim milk at room temperature for 1 h and subsequently incubated at 4°C overnight with the following primary antibodies (all 1:1,000 dilution): Anti-p53 (cat. no. ab26; Abcam, Cambridge, UK), anti-p21 (cat. no. ab109199; Abcam), anti-cleaved caspase-3 (cat. no. 9661; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-cleaved caspase-9 (cat. no. 9509; Cell Signaling Technology, Inc.), anti-p16 (cat. no. ab189034; Abcam), anti-p27^KIP1 (cat. no. ab193379; Abcam), anti-ATM, (cat. no. ab78; Abcam), anti-H2AX (cat. no. 7631; Cell Signaling Technology, Inc.) anti- phosphorylated-H2AX (γH2AX; cat. no. 07-164, EMD Millipore) and anti-β-actin (cat. no. sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Following incubation with the primary antibodies, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) or goat anti-mouse (cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) immunoglobulin G secondary antibodies at room temperature for 1 h (1:3,000 dilution). The immunoreactivity was detected using an enhanced chemiluminescence detection kit (EMD Millipore). The densitometric analysis was performed using Quantity One software (version 4.6; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

mLFCs were treated with 3 µM BaA or BaP for 72 h. Cellular senescence was analyzed by measuring the activity of SA-β-Gal. mLFCs were collected and fixed with 3% formaldehyde (Beijing Chemical Works, Beijing, China) for 3 min at room temperature. After washing with PBS, cell senescence was determined with the Senescence β-Galactosidase Staining kit according to the manufacturer's protocol (Beyotime Institute of Biotechnology). Images of the cells were captured using a light microscope and cells positive for β-Gal staining were subsequently counted.

The relative average telomere length of mLFCs was measured using total genomic mouse DNA as template, using quantitative polymerase chain reaction (qPCR), as previously described by Callicott and Womack (21), with certain modifications. Total genomic DNA was extracted from control, and BaA- and BaP-treated mLFCs using the Qiagen DNeasy Blood & Tissue kit (Qiagen China Co., Ltd., Shanghai, China). The primers used for the analysis were: Forward, 5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′ and reverse, 5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′. Ribosomal protein lateral stalk subunit P0 (RPLP0) gene was used as the reference gene. The following primers were used to amplify RPLP0: Forward, 5′-ACTGGTCTAGGACCCGAGAAG-3′ and reverse, 5′-TCAATGGTGCCTCTGGAGATT-3′. The qPCR reaction mixture to investigate telomere length and RPLP0 comprised 12.5 µl 2× SYBR-Green master mix (Roche Applied Science, Penzberg, Germany), 2.5 µl of each primer at a concentration of 300 nM and 20 ng genomic DNA; double-distilled water was added to a total volume of 25 µl. The PCR thermocycling conditions conducted to determine telomere length were as follows: Initial denaturation at 95°C for 10 min, followed by 30 cycles of 95°C for 15 sec and 56°C for 1 min, and final extension at 72°C for 5 min (ABI 7500; Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR thermocycling conditions for RPLP0 detection were as follows: Initial denaturation at 95°C for 10 min, followed by 35 cycles of 95°C for 15 sec, 52°C for 20 sec and 72°C for 30 sec, and final extension at 72°C for 5 min. Telomere length was assessed by calculating the relative ratio between telomeres and RPLP0. The 2^−ΔΔCq quantification method (22) was used to quantify the qPCR results.

Total RNA was extracted from mLFCs using the Qiagen RNeasy Mini kit (Qiagen China Co., Ltd.). Total RNA (800 ng) was reverse transcribed to cDNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The qPCR reaction mixture contained: 2 µl cDNA, 4 µl 2X Taq PCR StarMix buffer (GenStar, Beijing, China) and 2 µl of each primer. The semi-quantitative PCR amplification was performed using the GeneQ cycler (Hangzhou Bori Technology Co., Ltd., Hangzhou, China) and the thermocycling conditions were as follows: Initial denaturation at 98°C for 3 min, followed by 26–32 cycles of 95°C for 15 sec, 62°C for 15 sec and 72°C for 15 sec, and a final extension step at 72°C for 5 min. The qPCR reaction was performed as follows: Initial denaturation at 95°C 10 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. The primer sequences used were as follows: p16, forward, 5′-GAGCCATCTGGAGCAGCATGGAG-3′ and reverse, 5′-GCCCATCATCATCACCTGAATC-3′; p27^KIP1, forward, 5′-TGGGTTAGCGGAGCAGTGTC-3′ and reverse, 5′-CTCCACAGTGCCAGCGTTC-3′; and β-actin, forward, 5′-CAGGTCATCACTATTGGCAACGAGC-3′ and reverse, 5′-CGGATGTCAACGTCACACTTCATGA-3′. For semi-quantitative PCR, the reaction products were separated on a 3% agarose gel and visualized by ethidium bromide. The densitometric analysis was performed using Quantity One software (version 4.6; Bio-Rad Laboratories, Inc.).

Mice were exposed to exhaust gas for 30 days, and the CpG methylation state of the promoter of p16 was analyzed. Genomic DNA was extracted from mouse lung tissues, and control and 3 µM BaP-treated mLFCs. In total, 1,000 ng was used for bisulfite conversion with the QIAamp DNA Mini kit (Qiagen China Co., Ltd.) according to the manufacturer's protocol. PCR was performed using PrimeSTAR^® HS DNA Polymerase mix (cat. no. R044A; Takara Bio, Inc., Otsu, Japan). PCR thermocycling conditions were as follows: Initial denaturation at 97°C for 10 min, followed by 35 cycles of 96°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec, and a final extension at 72°C for 5 min. The primers used for assessing DNA methylation were as follows: Forward, 5′-GAGTTATTTGGAGTAGTATGGAG-3′ and reverse, 5′-ACCCATCATCATCACCTAAATC-3′. The PCR products were separated on 2% agarose gel and purified with a DNA gel extraction kit (Tiangen Biotech Co., Ltd., Beijing, China). Subsequently, the PCR products were cloned into a CloneJET vector using a PCR Cloning kit (Thermo Fisher Scientific, Inc.) and subjected to Sanger sequencing (Sangon Biotech Co., Ltd., Shanghai, China) for analysis of the methylation state of the p16 promoter.

SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. All experiments were repeated at least three times, and data are presented as the means ± standard deviation. Statistical significance was determined using Student's t-test or one-way analysis of variance followed by Fisher's least significant difference post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Extracted tissues were cut and cultured for 10 days, after which, mLFCs began to proliferate (Fig. 1G). After 7 additional days, mLFCs were trypsinized and transferred to a tissue culture plate for subsequent experiments (Fig. 1H).

mLFCs were treated with 3 µM BaA, BaP or BEP for 72 h, and cell proliferation was assessed using the WST-1 assay. mLFC proliferation was significantly suppressed following treatment with BaP, and proliferation was decreased by ~18% (P<0.01; Fig. 2A). Treatment with BaA decreased proliferation by 11%; however, the difference was not significant (P=0.13). Treatment with BEP did not alter cell proliferation. Cell apoptosis was subsequently determined by flow cytometry. Compared with the control group, cell apoptosis was increased by 7.8, 19.8 and 3.5% in the BaA, BaP and BEP groups, respectively (Fig. 2B). Treatment with BaP led to the highest apoptosis rate.

The protein expression levels of factors associated with cell growth and apoptosis were analyzed by western blotting (Fig. 3A). The results revealed that following treatment with 3 µM BaA or BaP, the protein expression levels of p53 and p21 were significantly increased. The protein expression levels of p53 and p21 were increased 1.7- and 2.7-fold in the BaA group, respectively (Fig. 3B). Furthermore, following treatment with BaP, the protein expression levels of p53 and p21 increased 1.9- and 3.9-fold, respectively (Fig. 3B). Additionally, the caspase-dependent cell apoptosis pathway was activated. The protein expression levels of cleaved caspase-3 and cleaved caspase-9 were increased 4.2- and 5.4-fold in the BaA group, and 4.0- and 7.5-fold in the BaP group, respectively (P<0.01).

A previous study demonstrated that long-term exposure to polluted air may cause biological aging and age-associated diseases (9). In the present study, PAH was hypothesized to induce premature senescence. Therefore, mLFCs were exposed to PAH for 72 h and senescence was examined. mLFCs treated with PAH were positive for SA-β-Gal activity (Fig. 4A). Compared with the control group, the number of cells positive for SA-β-Gal in the BaA and BaP groups was increased 2.1- and 4.6-fold, respectively (P<0.01; Fig. 4A). To further investigate whether cell senescence was associated with a decrease in telomere lengths, the telomeric regions were investigated using qPCR. No significant differences in telomere lengths between the control group and the samples exposed to PAH were identified (P>0.05; Fig. 4B), suggesting that PAH may promote cell senescence via the environment stress-induced premature senescence (SIPS) pathway and not via the replicative senescence (RS) pathway (23,24).

The expression levels of two senescence-associated factors, p16 and p27, were subsequently quantified (25–27). BaP exhibted the most notable effect on the induction of premature senescence (Fig. 4A); therefore, it was selected as the pollutant for subsequent experiments. Using semi-quantitative PCR, RT-qPCR and western blotting, the mRNA and protein expression levels of p16 and p27 were revealed to be significantly increased following treatment with BaP (P>0.01, Fig. 4C-E).

Cellular senescence is associated with the expression levels of p16, which is regulated by the methylation state of its promoter region (28). Since the present results suggested that PAHs may promote premature senescence and activate p16 in mLFCs, the DNA methylation state of the promoter of p16 was investigated following long-term exposure to EG or treatment with BaP. However, DNA sequencing results suggested that the methylation levels were similar between the control and the experimental groups (Fig. 5). The present results suggested that the premature senescence and the increase in the expression levels of p16 induced by PAHs were not associated with epigenetic alterations.

A previous study reported that ATM is involved in senescence downstream to various stimuli, including hyperproliferation and DNA damage (29). In response to DNA damage, ATM induces H2AX phosphorylation at serine 139, resulting in the phosphorylation of H2AX and an increase of γH2AX foci at the DNA damage sites (30,31). In the present study, the ATM/H2AX pathway was activated following exposure to PAHs. Western blot analysis results suggested that exposure to EG and PAH induced a significant upregulation of ATM and γH2AX (Fig. 6). The protein expression levels of ATM were increased 2.5- and 3.6-fold in the GE group and PAH group, respectively. Furthermore, the protein expression levels of γH2AX were increased 1.5- and 2.0-fold in the GE group and PAH group, respectively.