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Introduction

In recent years, due to the development of the Chinese economy, the severity of problems associated with air pollution has increased. Vehicle exhaust gas (EG) serves a principal role in air pollution and may induce various age-associated disorders, including cognitive dysfunction (1,2), metabolic dysregulation (3,4), cardiovascular disease (5,6) and cancer (7,8). Numerous studies have investigated the mechanisms underlying EG-associated disorders; DNA damage, epigenetic alterations, inflammation and oxidative stress have been identified to serve a role in these disorders (9–11).

Among the various chemical compounds identified in vehicle EG, polycyclic aromatic hydrocarbons (PAHs) are a group of chemicals containing at least two fused benzene rings without heteroatoms (12). PAHs may be released as a result of an incomplete combustion of derivatives of coal, petroleum or organic polymers. However, in urban zones, PAHs are primarily released from the engines of vehicles, suggesting that vehicle EG is the major source of PAHs in air pollution (13). The estimated concentration of PAHs in EG ranges between 200 and 500 parts per million, according to our previous study (data not published). Inhalation of PAH-containing air may increase the risk of lung cancer in humans; Osgood et al (14) demonstrated that PAHs may induce inflammation and tumorigenesis in mouse lung cells by activating extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase (MAPK) and inflammatory-associated genes, including cyclooxygenase 2 and chemokine ligand 2. Eom et al (15) conducted a pilot nested case-control study to examine the effects of exposure to PAH on lung carcinogenesis and identified that oxidative stress induced by exposure to PAH may be an important risk factor for lung cancer development. Furthermore, Zhao et al (16) demonstrated that benzopyrene (BaP) may promote lung cancer cell metastasis by activating the tumor necrosis factor-α signaling pathway. Additionally, accumulating evidence demonstrated that PAH may induce the methylation of genes involved in the development of cancer; White et al (17) demonstrated that PAH exposure is associated with hypomethylation and hypermethylation of various promoter regions in breast cancer. Additionally, Kim et al (18) identified that lipophilic PAHs contribute to the pathogenesis of insulin resistance through methylation-mediated suppression of the insulin receptor substrate 2 gene.

Zhu et al (19) and our previous studies (data not published) identified three principal types of PAH in vehicle EG derived from a gasoline internal combustion engine: Benzanthracene (BaA), BaP and benzoperylene (BEP). Additionally, our previous studies identified that exposure to vehicle EG may immunocompromise BALB/C mice (data not published). However, whether exposure to vehicle EG, particularly PAHs, is able to induce cell senescence remains unknown.

In the present study, vehicle EG and pure PAHs were used to simulate polluted air, and the cellular events following long-term PAH exposure were investigated by analyzing mouse lung fibroblast cells (mLFCs). PAHs were revealed to induce apoptosis of mLFCs, promoting various apoptosis-associated factors. Notably, PAHs induced cell senescence in mLFCs by activating the ATM serine/threonine kinase (ATM)/H2A histone family member X (H2AX) pathway. However, the epigenetic status of the promoter of senescence-associated genes, including p16, was not affected by exposure to PAHs. The present study may provide novel insights into the underlying mechanism of vehicle EG and PAHs in promoting the development of age-associated diseases.

Materials and methods

Animals and exposure model

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).

Figure 1. Isolation of mLFCs and establishment of EG and polycyclic aromatic hydrocarbons exposure model. (A) Diagram of the exposure chamber, consisting of five principal components: 1, Gas collecting bag; 2, controlled electromechanical injection system; 3, inhalation chamber; 4, air-mixing fan; 5, mouse cage. (B and C) BALB/C mice were exposed to EG for 30 days and sacrificed. Lung tissues were collected for molecular experiments. (D-F) Mice were sacrificed, lung tissues were collected and mLFCs were isolated. (G-I) mLFCs were culture-expanded and used for subsequent experiments. (G) Magnification, ×4; (H) magnification, ×20. Red arrow, mouse lung tissue; orange arrows, mLFCs. EG, exhaust gas; d, days; mLFC, mouse lung fibroblast cell.

Cell culture

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).

Cell proliferation assay

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: ODfinal=OD450-OD630-ODblank.

Cell apoptosis

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).

Protein extraction and western blotting

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-p27KIP1 (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).

Senescence-associated β-galactosidase (SA-β-Gal) staining

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.

Detection of telomere length in mLFCs

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.

Detection of gene expression levels by reverse transcription-qPCR (RT-qPCR) and semi-quantitative PCR

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′; p27KIP1, 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.).

DNA methylation analysis

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.

Statistical analysis

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.

Results

mLFC harvesting

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).

PAH induces mLFC apoptosis

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.

Figure 2. PAHs suppress proliferation and induce apoptosis of mLFCs. (A) mLFC proliferation was significantly decreased following exposure to BaP. (B) Flow cytometry and quantification of apoptosis rate suggested that PAHs may induce cell apoptosis. **P<0.01 vs. the CT group. BaA, benzanthracene; BaP, benzopyrene; BEP, benzoperylene; CT, control; mLFC, mouse lung fibroblast cell; NS, not significant; PAHs, polycyclic aromatic hydrocarbons; PI, propidium iodide.

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).

Figure 3. Exposure to BaA and BaP promotes the protein expression levels of factors associated with cell proliferation and apoptosis. (A) Protein expression levels of apoptosis-associated factors were detected by western blotting. (B) Densitometric analysis. **P<0.01 vs. the CT group. BaA, benzanthracene; BaP, benzopyrene; casp, caspase; CT, control.

PAH induces premature senescence in mLFCs

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).

Figure 4. PAH exposure induces premature senescence in mLFCs. (A) Cellular senescence was determined by senescence-associated β-Gal staining. (B) Relative telomere length of mLFCs was measured by qPCR. (C) mRNA expression levels of p16 and p27 were determined by semi-quantitative PCR (inverted color). (D) mRNA expression levels of p16 and p27 were determined by reverse transcription-qPCR. (E) Protein expression levels of p16 and p27 in mLFCs exposed to PAHs were determined by western blotting. **P<0.01 vs. the CT group; #P<0.01 vs. the BaA group. β-Gal, β-galactosidase; BaA, benzanthracene; BaP, benzopyrene; CT, control; M, marker; mLFC, mouse lung fibroblast cell; NS, not significant; qPCR, quantitative polymerase chain reaction; T/A, the ratio between the copies of telomere and the copies of the reference gene.

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).

PAH does not influence the DNA methylation state of the p16 promoter

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.

Figure 5. DNA methylation analysis of the p16 promoter. (A) Schematic diagram of the CpG islands on the p16 promoter. (B) p16 DNA methylation state of normal mLFCs. (C) p16 DNA methylation state of lung tissue following exposure to exhaust gas for 30 days. (D) p16 DNA methylation state of mLFCs following treatment with 3 µM BaP for 72 h. (E) Quantification of the total methylation rate in the three groups. White circles represent unmethylated CpGs, black circles represent methylated CpGs. BaP, benzopyrene; CT, control; GE, gas exposure; mLFC, mouse lung fibroblast cell; MR, methylation rate; NS, not significant.

PAH exposure activates the ATM/H2AX pathway

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.

Figure 6. GE and BaP exposure activates the ATM/H2AX pathway in mouse lung tissues and lung fibroblast cells. Mice were exposed to EG or clean air for 30 days and sacrificed, and the lung tissues were collected and protein was extracted. mLFCs were treated with 3 µM BaP for 72 h and the protein was also extracted. (A) Protein expression levels of ATM assessed by western blotting. (B) Densitometric analysis of the protein expression levels of ATM. (C) Protein expression levels of H2AX and γH2AX assessed by western blotting. (D) Densitometric analysis of the protein expression levels of γH2AX. The ratio between H2AX and γH2AX was determined, and expression levels were normalized to β-actin. (E) Diagram illustrating polycyclic aromatic hydrocarbons-promoted stress-induced premature senescence. **P<0.01 compared with the normal or control group. γH2AX, phosphorylated-H2AX; ATM, ATM serine/threonine kinase; BaA, benzanthracene; BaP, benzopyrene; BEP, benzoperylene; CT, control mLFCs; EG, exhaust gas; GE, gas exposure; H2AX, H2A histone family member X; mLFC, mouse lung fibroblast cell; NOR, normal (clean air).

Discussion

Vehicle EG contains various chemical compounds including carbon dioxide, hydrocarbons and nitrogen oxide, and it is important to investigate the biological effects of these components. PAHs are a group of toxic pollutants present in vehicle EG. Although PAHs are involved in the pathological progression of various tumors (32), the role of PAHs in cell senescence remains unclear. In the present study, under laboratory conditions, the biological effects of PAHs were investigated on mouse lungs and mLFCs. Cell proliferation was examined following exposure to PAHs for 72 h, and BaP, a type of PAH, significantly suppressed proliferation of mLFCs. Additionally, BaA and BaP were able to induce apoptosis of mLFCs. The molecular factors associated with apoptosis, including p53, p21, cleaved caspase-3 and cleaved caspase-9, were significantly upregulated following treatment with BaA and BaP. BEP did not significantly alter cell proliferation compared with the control, and exhibited a markedly reduced effect on apoptosis compared with BaA and BaP. Therefore, BaA and BaP were selected for further molecular experiments. A previous study suggested that the cytotoxic effects of BaA, BaP and BEP are distinct due to their differential potential to activate the aryl hydrocarbon receptor signaling pathway (33).

Two types of pathways may regulate cellular senescence: RS and SIPS. RS is induced by serial passage of normal cells in culture, whereas SIPS is primarily induced by exposure to environmental stimuli, including radiation and chemical toxicants (24). RS is associated with telomere shortening and epigenetic alterations, whereas SIPS is associated with DNA damage and genetic mutations. In the present study, it was demonstrated that BaA and BaP may promote cell senescence by increasing the expression levels of the senescence-associated factors p16 and p27 in mLFCs. Since telomere shortening is an important marker of RS (34), the lengths of telomeres were investigated in the PAH exposure group and in the control group. However, no significant difference between the two groups was observed.

PAHs may form reactive epoxides in cells, covalently binding to the DNA. These epoxides may induce epigenetic alterations, including cytosine methylation (35). Methylation of the p16 promoter is used as a marker of genomic hypermethylation (36). Furthermore, the hypermethylation of CpG islands in the p16 promoter has been identified to be an early event in lung cancer development; particularly in patients with a history of exposure to cigarette smoke (37). In the present study, using the bisulfite sequencing method, it was suggested that exposure to EG and PAH was not sufficient to affect the methylation status of the p16 promoter. Therefore, these present results, in combination with the results of the telomere length assay, suggested that PAH may induce SIPS and not RS.

Since DNA damage is a marker of SIPS, activation of the ATM/H2AX pathway was examined. The ATM/H2AX pathway is activated by DNA damage and regulates DNA repair (38). The present western blotting results suggested that exposure to EG and PAH upregulated the protein expression levels of ATM and γH2AX in mLFCs. A previous study demonstrated that the reactive oxygen species formed following exposure to BaP generate detrimental oxidative effects on cell proliferation and cell survival via an increase in membrane lipid peroxidation and oxidative DNA damage (16). Barascu et al (29) reported that oxidative stress may induce an ATM-independent senescence response via the p38 MAPK pathway. Therefore, SIPS induced by PAHs in vivo and in vitro may be associated with the oxidative stress-induced DNA damage response.

Collectively, the present study suggested that exposure to PAH may induce apoptosis of mLFCs and increase the protein expression levels of various apoptosis-associated factors, including p53, p21, caspase-3 and caspase-9. Additionally, exposure to PAH was revealed to generate a SIPS response in mLFCs, upregulating the expression levels of p16 and p27. Exposure to PAH did not influence the epigenetic status of the promoter of p16; however, PAH was identified to induce SIPS via activation of the ATM pathway, which may be initiated by reactive epoxides and oxidative effects of PAHs (Fig. 6E). The present study may provide novel insights into the underlying mechanism of vehicle EG and PAHs in promoting the development of age-associated diseases.

Acknowledgεments

Not applicable.

Funding

The present study was supported financially by The National Natural Science Foundation of China (grant no. 51306070).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

DQ contributed to the design of the study and wrote the manuscript. FY and KY performed the experiments and analyzed the data. YH contributed to the design of the study. JL was involved in conducting the experiments. YA analyzed data, providing constructive comments. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All animal experiments were approved by The Animal Care and Use and Ethics Committee on The Use of Animals of Jilin University.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References