Mutation Research 761 (2014) 35–43

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The apoptotic pathways effect of fine particulate from cooking oil fumes in primary fetal alveolar type II epithelial cells Zhen Che 1 , Ying Liu 1 , Yanyan Chen, Jiyu Cao ∗ , Chunmei Liang, Lei Wang, Rui Ding School of Public Health, Anhui Medical University, Hefei, China

a r t i c l e

i n f o

Article history: Received 7 January 2013 Received in revised form 19 December 2013 Accepted 7 January 2014 Available online 22 January 2014 Keywords: COF AEC II Apoptosis Caspase Cell cycles Flow cytometry

a b s t r a c t Apoptosis occurs along three major pathways: (i) an extrinsic pathway, mediated by death receptors; (ii) an intrinsic pathway centered on mitochondria; and (iii) an ER-stress pathway. We investigated the apoptotic pathway effects of cooking oil fumes (COF) in fetal lung type II-like epithelium cells (AEC II). Exposure to COF caused up-regulation of the pro-apoptotic protein Bax and down-regulation of the anti-apoptotic protein Bcl-2. COF induced the mitochondrial permeability transition, an early event in apoptosis; cytochrome c was translocated from the mitochondria to the cytoplasm and nucleus. Caspase9 and caspase-3 were activated, as a consequence of the mitochondrial permeability transition. The death receptor apoptotic pathway was triggered by COF, as indicated by a change in Fas expression, resulting in increased caspase-8 content. COF exposure arrested the cell cycle the at G0-G1 phase. In summary, COF can lead to apoptosis via mitochondrial and death receptor pathways in AEC II cells. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Epidemiological and toxicological studies have revealed that Chinese women exposed to cooking oil fumes have higher respiratory disease rates and mortalities due to lung cancer [1–4]. The strong negative health effects of exposure to the particles distributed in the cooking environment has been confirmed by Li et al. [5], Dennekamp et al. [6], Wallace et al. [7], See and Balasubramanian [8,9], and Varghese et al. [10]. These studies revealed that 60–90% of the particles associated with cooking are ultrafine particles that are produced with selected Western or Chinese cooking methods including frying, baking, deep-frying, and cooking of fatty foods and the cooking process could generate a large number of nanoparticles. Nemmar et al. [11] described that particulate matter can translocate from the lungs into circulation. Therefore, PM2.5 also induce inflammatory effects, as it has been shown in human epithelial lung cells [12]. Recent studies also have demonstrated that epithelial lung cells, macrophages, monocytes and granulocytes are susceptible to urban particulate matter by the induction of different cellular effects like oxidative stress, apoptosis, mitochondrial damage and NF-kB activation [13–17]. Because of some toxic components of PM2.5 are similar to that of urban particulate

∗ Corresponding author at: School of Public Health, Anhui Medical University, Meishan Road 81, Hefei 230032, Anhui Province, China. Tel.: +86 551 3869177. E-mail address: [email protected] (J. Cao). 1 These authors contributed equally to this work. 1383-5718/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrgentox.2014.01.004

matter, such as polycyclic aromatic hydrocarbons (PAHs) and aldehydes. Thus, we assumed that COF also can induce oxidative stress, apoptosis like urban particulate matter. It is well known that apoptosis and necrosis represent two distinct types of cell death. Apoptosis possesses unique morphological and biochemical features which distinguish this mechanism of programmed cell death from necrosis. Apoptosis is a cellular suicide or a programmed cell death which is mediated by the activation of an evolutionary conserved intracellular pathway. The relation of apoptosis and cancer has been emphasized and increasing evidence suggests that the process of neoplastic transformation, progression and metastasis involve alterations of normal apoptotic pathway [18]. Experimental observations [19–26] have shown that apoptosis occurs along three major pathways: (i) an extrinsic pathway mediated by death receptors, (ii) an intrinsic pathway centered on mitochondria, and (iii) an ER-stress pathway. Although many pathways of apoptosis may exist, only two main caspase cascades have been elucidated in detail in mammalian cells [27–29]. Mammalian cells have two main pathways that lead to apoptosis, namely, extrinsic and intrinsic pathways. The extrinsic is initiated by extrinsic ligand binding to death receptors, such as Fas (CD95), which in turn activates caspase-8 [30]. The intrinsic mitochondrial pathway is regulated by Bcl-2 family proteins [31,32]. Down regulation of Bcl-2 or Bcl-xL could result in permeabilization of mitochondrial outer membrane and facilitate the release of mitochondrial cytochrome c into cytoplasm [33]. Cytochrome c binds to Apaf1 and caspase-9 to form a multiprotein complex called apoptosome, which affects apoptotic process [34]. For the above mentioned

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reasons, the expressions of apoptotic proteins Bcl-2 family, Fas, caspase-3, caspase-8 and caspase-9 were analyzed with western blot for observing changes in apoptosis of mitochondrial and death receptor pathways in the AEC II cells exposed to COF. Cell cycle and apoptosis are important regulatory mechanisms of cell growth, development and differentiation. In mammals, the cell cycle comprises the G1, S, G2 and M phases. Cell cycle checkpoints ensure the maintenance of genomic integrity by inhibiting damaged or incomplete DNA. In this study, thus, we investigated the percentages of the AEC II cells exposed to COF in every phase using flow cytometry to understand cell cycle distribution. In this study, we have cultured primary mouse fetal lung alveolar epithelial type II cells successfully by differential adherence method and exposed the cells to extracts of PM2.5 in COF. This study further complements the studies on lesions of lung exposed to cooking oil fumes. We showed that COF may exhibit effective cell proliferation inhibition, partially by inducing mitochondrial and death receptor apoptotic pathways. 2. Materials and methods 2.1. Materials Microplate Reader (sunrise, Australia), inverted microscope (OLYMPUS), and JEM-1230 transmission Electron Microscope (Japan) were used in this study. The EP-13 PM2.5 sampler, filter papers were purchased from Nai Pu Electronic instrument (China) and the filter papers were made of ultra-fine fiber glass. TritonX-100, DMSO, Agarose, low melting point agarose and alkaline phosphatase, propidium iodide (PI) and RNase A were obtained from Sigma Chemical Co. (St. Louis. MO., USA). Anti-cytochrome c, -Bax, -BCL-2, -Fas, -FasL and caspase-3 (9662P; Caspase-3 Antibody detects endogenous levels of full length caspase-3 (35 kDa)), caspase-8, caspase-9 antibodies were from Cell Signaling Technology (Danvers, MA, USA). The Annexin V-FITC-based Apoptosis Detection Kit was purchased from BestBio company (Shanghai, China). Other chemicals and reagents were of analytical grade Peanut oil produced by hardpressing peanut kernels was purchased from a local market in Hefei, China. The peanuts had been roasted at 200 ◦ C for 45 min and precipitated at room temperature for 7–10 days, before the sediment was removed with filtration.

stain (10 mg naphthol-AS-bisphosphate [Sigma, N-5625] in 40 ml dimethylsuphoxide, made up to 10 ml with 0.625 M MgCl and 0.125 M amino methyl propanol in distilled water and filtered immediately before use) for 20 min at 30 ◦ C and washed with HBSS. Intense, pink staining identified the AEC II cells. 2.4.2. Electronic microscopy The AEC II cells were seeded into Petri dishes (6 and 10 cm) and exposed to 10 ␮g/ml (equates to 2.26 ␮g/cm2 ) COF for 1 h. Afterwards cells were trypsinized, resuspended in FBS-containing culture medium and centrifuged. Medium was then decanted, and the cells were washed twice with serum free medium and fixed with 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) for 1 h at 4 ◦ C. After incubation in 1% OsO4 in 0.1 PBS for 1 h at 4 ◦ C, the samples were washed in PBS, dehydrated in a graded series of acetone, and embedded in the epoxy resign Araldite. Ultrathin sections were cut with the Ultramicrotome Ultracut S, deposited on copper grids, stained with uranyl acetate and lead citrate and studied under a transmission electron microscope. 2.5. Cell treatment In the Western blotting assay, AEC II cells were seeded in a 6-well cell culture plate at a density of 2×105 cells/well. After 24 h of growth, the cells exposed to COF (0, 12.5, 25, 50 and 75 ␮g/ml), solution control (0.1% DMSO) and positive control (UV, 10 min)were dissolved in the cell culture medium (DMEM + 10% fetal bovine serum) and treated for 36 h. Flow cytometry was used to determine the cell cycle distribution and apoptosis induced by COF. In short, the AEC II cells were seeded in 100 mm culture dish at the density of 1 × 106/dish with 4 ml of DMEM supplemented with 10% FBS. Having been incubated overnight, the cells were treated with different doses of COF (0, 12.5, 25, 50 and 75 ␮g/ml) and incubated for 12, 24 and 36 h. 2.6. Apoptosis analysis Apoptotic cells were identified and quantified using the Annexin V-FITC-based Apoptosis Detection Kit, according to manufacturer’s instructions (BestBio, Shanghai, China). The AEC II cells were grown in multi-well plates, harvested by treatment with trypsin–EDTA and washed with cold PBS. Cells (1 × 106) were resuspended in 100 ␮l of 1× binding buffer supplemented with 5 ␮l of Annexin V-FITC and 5 ␮l of propidium iodide (PI), gently mixed and incubated at room temperature in the dark for 15 min. After the addition of 400 ␮l of 1× binding buffer, cells were kept on ice and immediately subjected to FACS analysis. At least 5000 events were recorded for each sample and represented as dot plots. Viable cells were negative for both PI and Annexin V, early apoptotic cells were positive for Annexin V and negative for PI, whereas dead cells displayed both Annexin V and PI labeling.

2.2. Collection and extraction of heated peanut oil fumes 2.7. Western blot analysis The collection and extraction of fumes were in accordance with the procedures of previous study [35]. Peanut oil (200 ml) was poured into an iron pot and heated with an electric heater. The temperature was maintained at the smoke point (280 ± 10 ◦ C). The fumes generated by the heating oil were collected, at a flow rate of 12 l/min, with the sampler placed 50 cm upon the oil surface which connected to a vacuum pump. Each filter paper was renewed after 1 h. The total amount of weight of fume particles collected from heated peanut oil for 1 h was 1.25 mg. The experiments were independently repeated three times. The gathered condensates were separately extracted with 200 ml of acetone using a Soxhlet extractor for 24 h. The extracts were then dried by rotary evaporation at 40 ◦ C. The eluant was evaporated to a yellow viscous solution and diluted to 200 mg/ml with dimethylsulfoxide (DMSO). The samples were sealed up in brown glass vials and preserved at −80 ◦ C until use.

After treatment with COF, the AEC II cells were lysed in cell lysis buffer (1% TritonX-100, 50 mM Tris-Cl pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na3 VO4 , 1 mM PMSF, and 2 mg/ml aprotinin) on ice, then centrifuged at 12,000 g for 10 min at 4 ◦ C. Protein concentration was measured by BCA protein concentration Kit, identical amounts (50 mg of protein) of cell lysates were boiled for 5 min and resolved by SDS-PAGE, then electrophoretically transferred onto a nitro-cellulose membrane, the membrane was blocked with 5% non-fat dry milk in Tris-BufferedSaline with Tween (TBST) overnight at 4 ◦ C, then incubated with appropriate primary antibodies in blocking buffer for 3 h at room temperature. After washed with TBST, the membrane was then incubated with appropriate secondary antibody for 2 h at room temperature. After extensive washing with TBST, proteins were visualized by the enhanced chemiluminescence’s (ECL) assay kit (Thermo Scientific, Rockford, USA).

2.3. Cell culture 2.8. Cell cycle distribution ICR mice were purchased from the Central Institute of Anhui Animals in Anhui, China. They were kept on a 12 h light/12 h dark cycle at a room temperature of 22 ± 2 ◦ C and free accessed to food and water. The mice were mated after an accommodation period of 1week. The pregnant mice were sacrificed through CO2 inhalation on the 18th day of gestation, and the fetuses were decapitated. Fetal lungs were rapidly removed under sterile conditions, and type II cells were isolated using the method of Batenburg et al. [36] with modifications. The cells were cultured in Dulbecco’s Modified Eagle Medium (containing 10% FBS, 100 units/ml of penicillin and100 units/ml of streptomycin, pH 7.4) with 95% air and 5% CO2 at 37 ◦ C. When the cells grew to 5–6 × 106 cells/ml, they were harvested with 0.25% trypsin in 0.02% EDTA and obtained after centrifugating at 1400 rpm for 5 min. Phosphate buffer saline (PBS) was added to adjust the cell number to 5 × 105 cells/ml for the following experiments and analyses.

Afterward cell treatment, the cells were trypsinized, pelleted, and resuspended at 5×106 cells/ml in PBS. The suspension was gently quivered, while three volumes of ice-cold 80% ethanol were added to it gradually. Fixed cells were stored at −20 ◦ C overnight and protected from light, until analysis. The cells in the solution were then pelleted and resuspended in equal volumes 30 ␮g/ml PI and 100 ␮g/ml Rnase A, both in PBS. Stained cells were incubated at 37 ◦ C for 30 min before analysis. The distributions of cells in the various cell cycle compartments were performed using a Becton Dickinson FacScan and appropriate software. For each sample, at least 5000 cells were analyzed. Data were collected for each analysis. Modeling of cell cycle stages was performed using ModFit software. Each treatment was tested in three individual dishes. 2.9. Statistical analysis

2.4. Characterization of human AEC II cells 2.4.1. Light microscopy The AEC II cell phenotype was identified by positive staining for alkaline phosphatase. Freshly washed, confluent cells were incubated with alkaline phosphatase

All data are presented as the means ± standard deviation (S.D.) (¯x ± s) Statistical analysis was performed using SPSS 11.0 software. Analyses of variances of multiple groups were performed using homogeneity test of variances and one-way ANOVA procedures. When the means were compared through pairwise comparison, the

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Fig. 1. Identification of the AEC II cells. Fetal lung type II-like epithelium cells were cultured in DMEM with 10% FBS. After 2 d, the cells were processed for light microscopy (A), phase microscopy (B), positive staining for alkaline phosphatase (C), and electron microscopy (D).

SNK test Student–Newman–Keuls method) was used for equal variance or GamesHowell was used. The levels of statistical significance employed were P < 0.05.

3. Results 3.1. Identification of AEC II The AEC II cells reached confluence between 2 and 3 d after plated. Using this method, confluent cells were positive for alkaline phosphatase. It was found that removal of monocytes and other cells, initially by saline perfusion and then by differential adhesion to tissue culture plastic before plating the remaining cells onto Vitrogen 100, consistently resulted in pure AEC II cell monolayer preparations, as described here. Nevertheless, cytospins of the initial preparation, immediately before plating, were only 60–80% positive for alkaline phosphatase, but alkaline phosphatasenegative cells did not adhere and were removed during the medium change at 36 h. They represented a highly differentiated pure cell type and could be grown in primary culture. The type II cells formed insula when cultured on Matrigel, as well described for mice type II cells (Fig. 1A and B). Intense, pink staining by alkaline phosphatase (Fig. 1C). The transmission electron microscopic image depicted that the AEC II cells maintained their morphological characteristics, which were lamellar bodies and high density of organelles, in primary culture for a couple of days (Fig. 1D). 3.2. Cells apoptosis studies To develop a model of apoptosis induced by COF in the AEC II cells, we treated the AEC II cells with COF. As shown in Fig. 2, treatment with COF resulted in a concentration- and time-dependent increase in the percentage of cells staining positive for Annexin V and negative for propidium iodide (PI), indicative of cells in the early phase of apoptosis. The results indicate that approximately 20–60% of cells were Annexin V+/PI− and/or Annexin V+/PI+ after

12 and 36 h of treatment with 12.5 ␮g/ml and 75 ␮g/ml COF. The percentage of cells in the early phase of apoptosis for treatment with 75 ␮g/ml COF after 36 h was 13.1% more than that for treatment with 75 ␮g/ml after 12 h which was 12.3%. And the percentage of cells treated with 12.5 ␮g/ml after 36 h and 12 h were 9.53 and 4.93%, respectively (Fig. 2). 3.3. Apoptosis pathways protein expression 3.3.1. The protein expressions of Fas, Caspase-3 and Caspase-8 The expressions of Fas, Caspase-8 and Caspase-3 were examined in the AEC II cells after being exposed to PM2.5 in COF but FasL was not detected. The values of Fas/␤-actin in 50 ␮g/ml and 75 ␮g/ml groups were higher (P < 0.05) than DMSO, 12.5 ␮g/ml and 25 ␮g/ml groups. Compared with DMSO and 12.5 ␮g/ml groups, there were statistically significant increases in the value of Caspase8/␤-actin in 50 ␮g/ml and 75 ␮g/ml COF treated groups (P < 0.05). In addition, the value of Caspase8/␤-actin in 75 ␮g/ml group was higher than that in 25 and 50 ␮g/ml groups. The values of Caspase3/␤-actin in 50 and 75 ␮g/ml groups were higher (P < 0.05) than DMSO, 12.5 and 25 ␮g/ml groups. According to above data, COF exposure induced increases in the protein expressions of Fas, Caspase-3, Caspase-8 in a concentration-dependent manner (Fig. 3). 3.3.2. The protein expression of Bax and Bcl-2 Compared with DMSO group, the protein expressions of Bax had statistically significant increases in a dose-dependent manner in the AEC II cells (P < 0.05). The value of Bcl-2/␤-actin in 12.5 ␮g/ml group was higher (P < 0.05) than that in DMSO group, but there were no statistically significant differences between other COF treated groups and DMSO group. The ratios of Bax/Bcl-2 increased statistically significantly with the concentrations of COF (P < 0.05) in a dose-dependent manner compared with that in DMSO group (Fig. 4).

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Fig. 2. Characterization of apoptosis changes in the AEC II cells. (A) Cell death was determined by Annexin V/PI staining and flow cytometry analysis at 12 h in cells treated with DMSO, 12.5 and 75 ␮g/ml and (B) 36 h in cells treated with DMSO, 12.5 and 75 ␮g/ml COF. The percent of the early phase apoptosis of cells increased significantly (P < 0.05) in a concentration- and time-dependent manner.

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Fig. 5. The protein expressions of Cyt-c, Caspase-9 and Caspase-3. Protein expression changes of Cyt-c, Caspase-9 and Caspase-3 in the AEC II cells in each exposure group for 36 h (concentration: ␮g/ml). According to data showed in Results, COF exposure induced increases in the protein expressions of Cyt-c, Caspase-9 and Caspase-3 in a concentration-dependent manner.

Fig. 3. The protein expressions of Fas, caspase-3 and caspase-8. Protein expression changes of death receptor signaling pathway in the AEC II cells in each group for 36 h (concentration: ␮g/ml). (A) Protein expression of protein Caspase-3 and Caspase-8, the values of caspase/␤-actin increased significantly (P < 0.05) in a concentrationdependent manner. (B) Protein expression of Fas, the value of Fas/␤-actin increased significantly (P < 0.05) in a concentration-dependent manner. According to data showed in Results, COF exposure induced increases in the protein expressions of Fas, Caspase-3, Caspase-8 in a concentration-dependent manner.

3.3.3. The protein expression of Cyt-c, Caspase-9 and Caspase-3 The value of Cyt-c/␤-actin in 12.5 ␮g/ml group was lower (P < 0.05) than that in DMSO group. And statistically significant increases in the values of Cyt-c/␤-actin were observed in the cells in 25, 50 and 75 ␮g/ml groups with a dose-dependent manner compared with that in 12.5 ␮g/ml group. The values of Caspase-9/␤-actin increased statistically significantly with the concentrations of COF (P < 0.05) in a dose-dependent manner compared with that in DMSO group. Moreover, 50 and 75 ␮g/ml groups particularly had statistically significant differences in the ratios of Caspase-9/␤-actin with DMSO group (P < 0.05). Compared with DMSO group, the values of Caspase-3/␤-actin increased statistically significantly with the concentrations of COF (P < 0.05) in a dose-dependent manner and there were statistically significant differences between 50 ␮g/ml, 75 ␮g/ml groups and DMSO group (P < 0.05) (Fig. 5). 3.4. Cell cycle distribution 3.4.1. Effects on cells in G0–G1 phase The effect of COF in cells was analyzed by flow cytometry for further exploring the observed effects of COF on cell cycle.

Fig. 4. The protein expressions of Bax and Bcl-2. Protein expression changes of Bax and Bcl-2 in the AEC II cells in each group for 36 h (concentration: ␮g/ml). The values of Bax/␤-actin increased significantly (P < 0.05) in a concentration-dependent manner. The ratios of Bax/Bcl-2 increased statistically significantly with the concentrations of COF (P < 0.05) in a concentration-dependent manner compared with that in DMSO group.

After the AEC II cells were exposed to different concentrations of COF for 12, 24 and 36 h, the percentages of these cells in G0–G1 phase were both higher than that in DMSO group at every period. The percentages of cells in G0–G1 phase statistically significantly increased by a dose-dependent manner were observed after the cells exposed to COF for 12 h and 36 h in 50 and 75 ␮g/ml COF treated groups (P < 0.05) compared to the percentages observed in DMSO and 12.5 ␮g/ml groups. Excepted for 12.5 ␮g/ml COF treated group (P > 0.05), there existed statistically significant increases in the percentages of cells in G0–G1 phase were observed after the cells exposed to COF for 24 h in all COF treated groups compared to the percentage observed in DMSO group. Furthermore, the percentages also increased in a dose-dependent manner (Fig. 6). 3.4.2. Effects on cells in S phase After the AEC II cells were exposed to different concentrations of PM2.5 at 12, 24 and 36 h, the percentages of these cells in S phase were both lower than that in DMSO group at every period. The percentages of cells in S phase statistically significantly decreased in a dose-dependent manner were observed after the cells exposed to COF for 12 and 36 h in every COF treated group (P < 0.05) compared to the percentages observed in DMSO group. Excepted for 12.5 ␮g/ml COF treated group (P > 0.05), there existed statistically significant decreases in the percentages of cells in S phase were observed after the cells exposed to COF for 24 h in all COF treated groups compared to the percentage observed in DMSO group. Moreover, the percentages also decreased in a dose-dependent manner (Fig. 6). 4. Discussion Lung, the essential respiratory organ of the body is an important target organ for inhaled poison. Alveolar is the basic functional unit of lung for gas exchange whose epithelial cells are formed by type I and type II epithelial cells. The type II alveolar epithelial cells cannot only synthesis and secrete pulmonary surfactant secretion, but also be differentiated into type I alveolar epithelial cells, which are considered as the stem cells of alveolar epithelial cells. The abnormalities of their proliferation, apoptosis and differentiation may have significant impacts on body. Oil prepared from roasted peanut contains a tasteful aroma and is popular in the orient. Unfortunately, this oil is regarded as a ‘fume maker’ because it has a smoke point around 100 ◦ C, which is 70 ◦ C lower than that of the soybean oil. Since the harms of the fumes from peanut oil are still unclear, peanut oil with a low smoke point temperature is a suitable material for investigating the mutagenicity and mutagens in oil fumes. COF contains two major classes of compounds. One class consists of polycyclic aromatic hydrocarbons

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Fig. 6. The flow cytometry graphic of the AEC II cells distribution. (A) The flow cytometry graphic of the distribution of AEC II cells exposed to PM2.5 for 12 h; (B) the distribution of AEC II cells exposed to PM2.5 for 24 h; (C) the distribution of AEC II cells exposed to PM2.5 for 36 h. The percentages of cells in G0–G1 phase statistically significantly increased in a dose-dependent manner were observed after the cells exposed to COF for 12, 24 and 36 h in 50 and 75 ␮g/ml COF treated groups (P < 0.05) compared to the percentages observed in DMSO group. And the percentages of cells in S phase statistically significantly decreased in a dose-dependent manner were observed after the cells exposed to COF for 12, 24 and 36 h (P < 0.05) compared to the percentages observed in DMSO group.

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(PAHs), such as benzo[a]pyrene, benzo[b]fluoranthene, fluoranthene, and benzo[g,h,i]perylene. Chiang et al. [37] successfully identified four PAHs in fumes from these cooking oils, BaP, DBahA, and BaA are considered probable human carcinogens (group 2A); BbFA is considered a possible human carcinogens (group 2B) by IARC (1992). For example, BaP is a known immunosuppressant. It can also alter cell cycle progression, induce inflammation, and impair DNA repair and apoptotic processes leading to aberrant cellular functioning. Recent reports also indicate that oil fumes resulting from heating edible oils to high temperature exhibit mutagenicity and genetic toxicity [38,39]. 12 major compounds were analyzed and confirmed with authentic compounds by Wu et al. [40] by means of GC/MS. Those 12 compounds, including three alcohols, five aldehydes, two dienals, and two alkanes, were 1-pentanol, 2-heptenal, nonanal, trans-2-octenal, n-heptanol, n-pentadecane, 1-octanol, trans-2-decenal, n-heptadecane, rans-trans-2,4nonadienal, trans-2-undecenal, and trans-trans-2,4-decadienal. trans-trans-2,4-decadiena l had the highest amount (51.6%), and the amounts of n-nonanal, trans-undecenal, and trans-2-decenal were 13.2, 10.4, and 7.2%, respectively. They are major products of this degradation and they are able to induce toxicological effects (e.g. their reactivity with amino groups of proteins), such as aldehydes are considered to have high relevance with genetic toxicity. We have cultured primary mouse fetal lung alveolar epithelial type II cells successfully by differential adherence method and exposed the cells to extracts of PM2.5 in COF. This study further complements the studies on lesions of lung exposed to cooking oil fumes. A cell cycle can be divided into four periods, namely, the G1 phase, S phase, G2 phase and M phase. The main activities of cells in the G1 phase are the synthesis of protein and RNA in order to replicate cytoplasm, while the synthesis of DNA occurs in S phase. G2-M phase is the period from the completion of DNA synthesis to the end of mitosis when cells are divided. The phenomenon that cells stay too long in G1 and G2 phases is called G1 and G2 arrest, this arrest is under control of a series of molecular mechanisms, which plays an crucial role in preventing from damaged DNA replication in the S phase or division in the M phase, avoiding the erroneous genetic information entering the progeny cells in M phase, maintaining the stability of the cells’ genetic properties. It is a protective effect of the body to respond to the external stimulations. G1 phase arrest may provide sufficient time to induce the repairment of damaged DNA, which mainly depends on the regulation of P53 protein. Damaged cells are eliminated by apoptosis when they are not repaired successfully. When some damaged DNA enter into the next phase of cell cycle from one phrase before repaired, this damage can be fixed down to result in genetic instability of genome and potential likelihood of death. In this study, we exposed the AEC II cells to the PM2.5 in COF and compared them with the control group. We found that the percentage of cells in G0/G1 phase increased while the percentage of cells in S phase decreased with the concentration of exposure increasing when observed 12, 24 and 36 h after exposure. This result suggests that PM2.5 in COF can induce cell cycle arrest at the G0/G1 phase by concentration dependent way, showing G1 arrest. It is probably because that the AEC II cells are damaged by PM2.5 in cooking fumes and they need sufficient time to repair DNA. Deng [41] discovered that PM2.5 in normal air and sandstorm air can increase the percentage of lung fibroblast cells in G2/M phase and decrease the percentage of lung fibroblast cells in G0/G1 and S phases. Gualtieri [42] study has shown that the percentage of BEAS-2B in G2/M phase increased after they were exposed to the PM2.5 in winter air of Milan. We believe that the reasons of different results may be different sources of PM2.5 and different cell models. Apoptosis (programmed cell death) plays a crucial role for cell homeostasis and differs from necrosis and is necessary for maintenance of development and homeostasis of multicellular organisms

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by eliminating superfluous or unwanted cells [43]. Inefficient apoptosis is considered as one of the hallmarks of tumorigenicity [44,45]. Among the morphological and biological changes that occur in apoptosis, the main manifestations of cell death are nuclear fragmentation, chromatin condensation, cell shrinkage and DNA fragmentation [46]. There are two major apoptosis signaling pathways. The death receptor (extrinsic) pathway and the mitochondria (intrinsic) mediated pathway. The extrinsic pathway is initiated by cell surface expressed death receptors of the tumor necrosis factor superfamily. One of the central pathways of apoptosis is initiated by cytokines, such as tumor necrosis factor-␣(TNF-␣), Fas ligand (FasL) and tumor necrosis factor-␣-related apoptosis-inducing ligand (TRAIL) [47]. Once the receptor is activated, for example by Fas ligand, receptors oligomerize, recruit intracellular adaptor proteins and form scaffolding complexes, while FADD is recruited for Fas signaling [48]. The complex recruits one or more members of the caspase family of cell death protease, classically caspase-8. Cleavage of caspase-8 leads to the formation of an active enzyme comprising p20 and p10 heterotetramer. This activated initiator caspase cleaves downstream effector caspases, in particular caspase-3. Caspase-3 then cleaves a large number of intracellular substrates [49]. In this study, we investigated the Fas/FasL system which is a key signaling transduction pathway of apoptosis in cells and tissues [50]. Ligation of Fas by agonistic antibody or its mature ligand induces receptor oligomerization and formation of death-inducing signaling complex (DISC), followed by activation of caspase-8, then further activating a series caspase cascades resulting in cell apoptotic death [50]. Our results showed that PM2.5 in COF results in a significant increase of Fas protein expression, and also revealed the degree of caspase-8 and caspase-3 activity were enhanced after the AEC II cells were treated with COF. The intrinsic pathway involving key mitochondrial events such as anti-apoptotic proteins Bcl-2, Bcl-xL and proapoptotic protein Bax, Bad and Bid regulating the release of cytochrome c from mitochondria to cytosol and activating the initiator caspases-9 and effector caspase-3 [51]. Caspase-3 is a vital member of the caspase family, a group of cysteine proteases that mediate apoptotic execution [52]. It can be activated by apoptotic signals from both death receptor and intracellular/mitochondrial pathways. Caspase3 functions as a major effector caspase by cleavage of numerous cell death substrates, to cellular dysfunction and destruction [52]. Our results showed that PM2.5 in COF give rise to a significant increase of Bax protein expression, and a decrease of Bcl-2, suggesting that changes in the ratio of pro-apoptotic and anti-apoptotic Bcl-2 family members might contribute to the apoptosis-promotion activity of COF. In addition, our data also revealed the degree of caspase-9 and caspase-3 activity after the AEC II cells were treated with COF. The other study reported that Bcl-2 family proteins regulate the activation of caspase-9 and caspase-3 during apoptotic cell death [53,54]. These mitochondrial apoptotic events play an important role in COF-mediated apoptosis. Epithelial cell apoptosis is considered to be the initial event in various lung disorders. In our experiments, we also observed that COF can cause apoptosis, which was indicated by changes in increased caspase protein levels in cultured the AEC II cells. Due to the caspase protein levels increased, the intrinsic and extrinsic apoptosis pathways must be involved. It is also noticeable that apoptotic cell death paralleled necrotic cell death, even though they both can lead to different consequences. In contrast to necrotic cells, apoptotic cells can be completely engulfed by surrounding cells. No inflammatory reaction is caused by apoptotic cells, because there is no rupture of the cell membrane [55] (if the engulfment is timely). Although apoptosis may not cause a prolonged inflammatory reaction, because of this efficient clearance, it does play a role in the

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