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Aroclor 1254 inhibits cell viability and induces apoptosis of human A549 lung cancer cells by 2+
modulating the intracellular Ca level and ROS production through the mitochondrial pathway a
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Yufan Zhong , Panpan Guo , Xiu Wang & Jing An
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Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai, P. R. China Published online: 01 Jun 2015.
To cite this article: Yufan Zhong, Panpan Guo, Xiu Wang & Jing An (2015) Aroclor 1254 inhibits cell viability and induces 2+
apoptosis of human A549 lung cancer cells by modulating the intracellular Ca level and ROS production through the mitochondrial pathway, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 50:8, 806-813, DOI: 10.1080/10934529.2015.1019797 To link to this article: http://dx.doi.org/10.1080/10934529.2015.1019797
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Journal of Environmental Science and Health, Part A (2015) 50, 806–813 Copyright © Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2015.1019797
Aroclor 1254 inhibits cell viability and induces apoptosis of human A549 lung cancer cells by modulating the intracellular Ca2C level and ROS production through the mitochondrial pathway YUFANG ZHONG, PANPAN GUO, XIU WANG and JING AN
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Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai, P. R. China
To study the acute toxic effects of PCBs on airway exposure, the cell viability, apoptosis and mitochondrial functions of human lung cancer cell line A549 were measured and compared after Aroclor 1254 exposure for different time. The results showed that Aroclor 1254 could inhibit cell viability and increase cell apoptosis in a concentration- and time-dependent manner. The mitochondrial apoptosis pathway was confirmed playing an important role. ROS elevation was an early response within 1h treatment of Aroclor 1254. Then after 4 h of Aroclor 1254 exposure, the intracellular calcium level increased and mitochondrial transmembrane potential (DCm) collapsed, accompanying with Cytochrome c (Cyt-c) leakage, boosting expression of Bax, Apaf-1 and miRNA155, which were involved in the mitochondrial apoptosis pathway. After 24 h of Aroclor 1254 exposure, ROS returned to normal level, but cell apoptosis rate was higher than that at 4 h with DCm continued collapsing and intracellular calcium increased. In conclusion, Aroclor 1254 could suppress cell viability and induce apoptosis in A549 cells, which was associated with ROS over-production and elevated cellular Ca2C level, which may result in mitochondrial dysfunction, inducing expression of Bax/Cyt-c/Apaf-1 and miRNA155. Keywords: Aroclor 1254, A549, ROS, cell apoptosis, mitochondrial membrane potential, cellular Ca2C.
Introduction Polychlorinated biphenyls (PCBs) refer to a family of synthetic organic chemicals known as chlorinated hydrocarbons. PCBs had been widely used in electrical equipment, heat transfer, hydraulic equipment, and many other industrial applications as insulating additives before their ban in 1970s. However, PCBs continue to be released from the PCB-containing consumer products due to improper disposal, and enter environmental media including air, water, and soil. Due to their long half-lives, extreme persistence in environment and bio-accumulation capacity, PCBs exposure are thought to be a significant health hazard for human populations. Chronic exposure to PCBs and their metabolites have multifaceted adverse effects on wild organisms such as developmental deformities,[1,2] immunological defects,[3]
Address correspondence to Jing An, Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, P. R. China; E-mail: [email protected] Received October 27, 2014.
endocrine disruption[4,5] and potential carcinogenesis.[6,7] Results of animal studies and epidemiological studies showed that PCBs could cause cancer development and enhance metastatic propensity in various target organs including liver, brain, breast, and prostate.[8–10] PCBs can be bioaccumulated in the food web, and digestion of contaminated animal foods is regarded as a primary exposure route of PCBs.[11, 12] Some evidences have shown that inhalation uptake is also a significant source route of PCBs exposure.[13,14] Results of daily intake estimation for e-waste recycling workers indicate that air inhalation contributes a large portion of low-chlorinated PCB intake (>80% for triCBs).[15] In urban areas, inhalation of indoor dust and air has been highlighted as an important pathway of PCBs exposure.[16] Recent epidemiologic study found that children living near a hazardous PCBs waste site had higher risks of respiratory diseases.[17] Moreover, several PCB mixtures and congeners were reported to promote lung tumors in a mice model.[18,19] Therefore, assessment of health risk and toxicity mechanisms associated with exposure of airborne PCBs is necessary.
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Aroclor 1254 induces apoptosis of human A549 lung cancer cells Reactive oxygen species (ROS) are well-established inducers of oxidative damage responsible for various biological processes, such as cell viability, proliferation and cell death.[20-23] In the biological systems, ROS are usually used to assess toxic effects resulting from exposure to chemical toxins.[24] Considerable evidence showed that PCBs exposure could cause uncontrolled ROS accumulation and intracellular oxidative stress,[25,26] and eventually lead to various adverse disorders including respiratory system diseases.[27] Aroclor 1254 is a widely used commercial PCBs mixture containing dioxin-like and non-dioxin-like PCBs, as well as several highly potent polychlorinated dibenzo-p-dioxin and -furan impurities. Mitochondrion is susceptive target for chemicals exposure and a hinge of various cytotoxic progresses such as apoptosis. Bax, Cyt-c and Apaf-1 play important role in the mitochondria involved apoptosis. In this study, the toxic effects of Aroclor 1254 in the human lung carcinoma cell line (A549) were evaluated through measurement of cell viability, apoptosis, ROS production, intracellular Ca2C Level, mitochondrial membrane potential (DCm) and Cyt-c release. In addition, expression of miRNA155, Apaf-1 and Bax in A549 cells exposed to Aroclor 1254 was determined to explore the underlying molecular mechanism.
Materials and methods Materials A549 cell line was from American Type Culture Collection (ATCC; Manassas, VA, USA). The Ham’s F-12 culture media and fetal bovine serum (FBS) were purchased from GE (Piscataway, NJ, USA). Aroclor 1254 (CAS no: 11097-69-1) was purchased from AccuStandard (New Haven, CT, USA). Solutions of Aroclor 1254 at strength of 20 mg/mL were prepared in DMSO and diluted as necessary with the cell culture medium. Other chemicals and kits were purchased from the companies as following: Cell Counting Kit-8 (CCK-8), Fluo-4 AM and F-127 from Dojindo (Kumamoto, Japan); Nuclear and Cytoplasmic Extraction Reagent Kit (NE-PER), Mammalian Protein Extraction Reagent (M-PER) and BCA kit from Thermo Fisher Scientific Inc (Waltham, MA, USA); Apoptosis detection kit from Mbchem (Shanghai, China); 20 ,70 dichlorofluorescein diacetate (DCFH-DA), tert-Butyl hydroperoxide (tBHP) and PVDF membrane from Millipore (Darmstadt, Germany); TRIzol from Invitrogen (Paisley, UK); SYBR Green qPCR Master Mix Kit and Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase from TOYOBO (Osaka, Japan); Rabbit monoclonal antibodies for Cytochrome C (Cyt-c), Apaf-1 and Bax from Epitomics (Burlingame, CA, USA), mouse polyclonal antibody for GAPDH from Multisciences Biotechnology (Hangzhou, China); anti-Rabbit IgG(HCL)/HRP
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and anti-Mouse IgG (HCL)/HRP from Dingguo Biotechnology (Beijing, China); Rhodamine 123, DMSO and other analytical grade reagents from Sigma (St. Louis, MO, USA). Absorbance was recorded using iMark plate reader (BioRad, Hercules, CA, USA). Apoptosis was assayed by flow cytometry (Becton Dickinson, San Jose, CA, USA) and data was analyzed using Cell Quest Software (Becton Dickinson, San Jose, CA, USA). Fluorescence was detected by fluorescence microscope (Olympus BX-51, Tokyo, Japan) and analyzed by Image-pro Plus 6.0 software (IPP, Media Cybernetics, Rockville, MD, USA). Quantitative PCR ran in ABI Prism 7500 Sequence Detection system (Applied Biosystems, Waltham, MA, USA). The optical densities of western blotting bands were quantized using the Chemi-Imager digital imaging system (Alpha Innotech, San Leandro, CA, USA). Cell culture and treatments A549 cells was cultured in 10% complete medium (Ham’s F-12 medium, 10% fetal bovine serum, 2 mM glutamine, 0.33% sodium bicarbonate, 100 units mL¡1 penicillin and 0.1 mg/mL streptomycin) in humidified incubator with 5% CO2 at 37℃. Before Aroclor 1254 treatment, A549 cells at exponentially growing phase were seeded in culture plates and grown for 24 h to 70–80% confluence. Aroclor 1254 were dissolved in DMSO and diluted with culture media to 0, 1, 5 and 10 mg/mL with a final concentration of DMSO at 0.1% v/v. After Aroclor 1254 treatment (1 h, 4 h or 24 h), cells were washed with D-Hanks and subjected to following assay procedures. All experiments were carried out in at least triplicate, and 3 parallel samples were tested for each concentration (6 parallel samples were applied in cck-8 test). Cell viability First, 5 £ 103 cells well¡1 seeded in 96-well plates were treated with different concentrations of Aroclor 1254 (0, 1, 5, 10 mg mL¡1) for 24 h, and cell viability was measured by Cell Counting kit-8 (CCK-8) as the description of kit. In brief, 10 mL of CCK-8 reagent in 90 mL Ham’s F-12 medium per well was added to cell culture medium, and then incubated at 37℃ for 1 h. Absorbance of each well was measured at 450 nm with an iMark plate reader. Assessment of apoptosis Apoptosis of A549 cells was measured using flow cytometry with Annexin V-FITC/PI double staining assay kit. After treatment with various concentrations of Aroclor 1254, cells were dispersed with 0.25% trypsin, then washed twice with D-Hank’s and suspended at a concentration of 1 £ 106 cells mL¡1 by 1 £ binding buffer (10 mM
808 HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). After 30 min incubation at 37℃ in darkness with Annexin V-FITC and propidium iodide (PI), cells were immediately analyzed by flow cytometry using the FL2 detector at 488 nm excitation wavelength.
Yufan et al. amounts of protein (60 mg per lane) were subjected to 10% SDS-PAGE separation and transferred (semidry transfer assay) from the gel onto an ethanol saturated PVDF membrane, which was then blocked by BSA and incubated with primary antibodies overnight at 4℃, blotted with secondary antibodies for 1 h at room temperature. Finally blots were visualized using chemiluminescence assay.
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Measurement of reactive oxygen species (ROS) When combined with the hydrogen peroxide, ROS probe DCFH-DA will convert to DCF, which emits bright green fluorescence. To evaluate the intracellular ROS generation induced by Aroclor 1254, 1 £ 106 cells per dish seeded in 35-mm culture dishes were treated with Aroclor 1254, then cells were washed twice with D-Hanks and loaded with 10 mM DCFH-DA at 37℃ for 30 min in darkness. Finally, cells were washed three times again with D-Hanks, and DCF fluorescence was measured by fluorescence microscope. Measurement of mitochondrial membrane potential (DCm) Rhodamine 123 (Rh123) is a membrane permeable fluorescent cationic dye, which is selectively taken up by mitochondria proportional to the Mitochondrial membrane potential. A549 cells cultured in 35-mm culture dishes were treated with different concentrations of Aroclor 1254 (0, 1, 5, 10 mg mL¡1) for 1, 4 or 24 h, then cells were washed twice with D-Hanks and incubated with 10 mg mL¡1 Rh123 at 37℃ for 30 min in darkness. Finally, cells were washed three times and fluorescence intensity was measured under fluorescence microscope. Measurement of intracellular Ca2C level Intracellular calcium levels were determined by ratiometric fluorimetry with the fluorescent calcium indicator Fluo-4 AM. After Aroclor 1254 treatments, approximately 1 £ 106 of A549 cells were seeded on 35-mm culture dishes, and rinsed 3 times with D-Hanks. Then cells were mixed with 4 mM Fluo-4 AM in the buffer for 30 min at 37℃, followed with washing three times with D-Hanks. Calcium imaging was conducted with fluorescence microscope at 488 nm wavelength. In this process, F-127 (0.05%, W/V) was used to prevent Fluo-4 AM from polymerization in the D-Hank’s solution. Western blotting For every sample, 2 £ 107 cells were lysed using the NEPER Kit or M-PER Kit to obtain cytoplasmic extracts or cell total protein respectively. The cytoplasmic extracts were used to measure the leakage of Cyt-c, and cell total protein was used to measure the expression of Apaf-1 and Bax. GAPDH was used as the internal reference. Equal
Real-time PCR Total RNA of A549 cells was extracted using TRIzol (1 £ 106 cells mL¡1), then reversely transcribed to cDNA using M-MLV reverse transcriptase. Approximately 1 mg cDNA for each sample was amplified in a 20-mL reaction mixture, which contains SYBR Green PCR Master Mix, appropriate primer pairs and nuclease-free water. The qPCR cycling conditions were as follow: 40 cycles of 94℃ for 10 s, 59℃ for 20 s, and 72℃ for 10 s. All samples were measured in triplicate with the human housekeeping gene b-actin being used as a reference to normalize data. The qPCR primer sets were listed as follows: miRNA155, 50 CTCTAATGGTGGCACAAA-30 (forward), and 50 TGATAAAAACAAACATGGGCTTGAC-30 (reverse); Apaf-1, 50 -GGAGGAC- CCTCAAGAGGATATG-30 (forward) and 50 -GGATTTCTCCCAATAG- GCCACT30 (reverse); b-actin, 50 -CCATGGAGAAGGCTGGGG-30 (forward) and 50 -CAAAGTGTCATG- GATGACC-30 (reverse). Statistical analyses All experiments were conducted in at least triplicate repeated wells for each sample, and data were presented as mean § standard deviation derived from three independent experiments. Statistical differences were conducted using SPSS 11.0 software (SPSS Inc., Chicago, IL, USA). The differences between control and exposed groups were evaluated by ANOVA. P values < 0.05 were considered to be statistically significant.
Results Aroclor 1254 could inhibit cell viability and increase apoptosis of A549 cells A549 cells were exposed to different concentrations of Aroclor 1254 and CCK-8 assay was conducted to determine the cell viability. As shown in Figure 1A, after Aroclor 1254 treatment, the cell viability obviously decreased in a concentration- and time-dependent manner (P < 0.05). Compared with the control group, the cell viability of 10 mg mL¡1 Aroclor 1254 groups reduced to 78% after 4 h treatment, 5 and 10 mg/mL groups significantly reduced to 49% and 24% after
Aroclor 1254 induces apoptosis of human A549 lung cancer cells
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Aroclor 1254 increased ROS production Intracellular ROS production was measured by DCFHDA assay. As shown in Figure 2, after Aroclor 1254 treatment for 1 h, the DCFH-DA fluorescence density of A549 cells was significantly (P < 0.01) elevated as compared with the control cells in a dose-dependent manner. After treatment for 4 h, Aroclor 1254 induced ROS elevation in all three treatment groups, while the increasing trend slowed (P < 0.05); and after 24 h treatment, the ROS level difference between control group and treatment groups disappeared (P > 0.05).
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Effects of Aroclor 1254 on mitochondrial membrane potential and intracellular Ca2C level
Fig. 1. The cell survival and apoptosis of A549 after treatment with Aroclor 1254 (A): The cell survival of A549 after treatment with different concentration (0, 1, 5 and 10 mg mL¡1) of Aroclor 1254 for1, 4, 24 h. CCK8 assay was used to evaluate the cell survival. (B): The apoptosis of A549 after treatment with different concentration (0, 1, 5 and 10 mg mL¡1) of Aroclor 1254 for1, 4, 24 h. The cell apoptosis was assessed by Annexin V-FITC/PI double staining assay. *P < 0.05, **P < 0.01 compared with the control group.
The Dcm was measured with Rhodamine 123 (Rh123) assay results (Fig. 3A) showed that Aroclor 1254 treatment induced loss of Dcm in a dose-dependent manner. In groups treated with 10 mg mL¡1 of Aroclor 1254, the Dcm significantly decreased compared with that of control group at 4 h or 24 h (P < 0.05); for 5 mg mL¡1 group, the Dcm loss appeared only at 24 h. In the 1 mg mL¡1 group,
24 h, respectively. Annexin V-FITC/PI double staining assay results (Fig. 1B) showed that Aroclor 1254 induced cell apoptosis in a time- and concentrationdependent manner. After Aroclor 1254 treatment, the apoptotic cell percentage of 5 and 10 mg mL¡1 groups was significantly higher than that of the control group; and the apoptosis rate at 24 h were even higher than that at 4 h both in 5 and 10 mg mL¡1 groups (Fig. 1).
Fig. 2. The ROS production induced by Aroclor 1254 treatment. A549 cells were treated with Aroclor 1254 (0, 1, 5 and 10 mg mL¡1) for 1, 4 or 24 h. Then the intracellular ROS level measured with DCFH-DA assay. *P < 0.05, **P < 0.01 compared with the control group.
Fig. 3. The Mitochondrial Membrane Potential and cellular Ca2C level of A549 after treatment with Aroclor 1254: (A): The Mitochondrial Membrane Potential of A549 after treatment of different concentration (0, 1, 5 and 10 mg mL¡1) of Aroclor 1254 for 1, 4 or 24 h. Rhodamine 123 (Rh123) assay was used to evaluate the Mitochondrial Membrane Potential. (B): The cellular Ca2C Level of A549 after treatment of different concentration (0, 1, 5 and 10 mg mL¡1) of Aroclor 1254 for 1, 4 or 24 h. For calcium measurement, cells after Aroclor 1254 treatment were incubated with Fluo 4-AM. Fluorescence images were recorded with a fluorescence microscope. *P < 0.05, **P < 0.01 compared with the control group.
810 no obvious Dcm decrease was observed. Measurement of Ca2C level was conducted with Fluo-4 AM/F-127 assay and the intensity of fluorescence was analyzed by Image-pro plus 6.0 software. Figure 3B showed that 5 and 10 mg mL¡1 Aroclor 1254 induced significant elevation of Ca2C level at 4 to 24 h (P < 0.05), while 1 mg mL¡1 group caused Ca2C level elevation only at 24 h (P < 0.05).
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Expression of apoptosis-related proteins induced by Aroclor 1254 Western blotting results showed that expression level of Bax and Apaf-1 (Fig. 4A) increased after Aroclor 1254 treatment, which peaked at 4 h, and recovered after 24 h treatment. In addition, the cytoplasmic Cyt-c (Fig. 4B) also increased after Aroclor 1254 treatment for 1 h, reached peak at 4 h, and then attenuated. Consistent with the Western blotting results, the real-time
Yufan et al. Table 1. The expression level of Apaf-1 and miRNA155 in A549 cells after Aroclor 1254 treatment. Groups Apaf-1 miRNA155
1h
4h
24 h
1.05 § 0.06 1.45 § 0.15
3.23 § 0.32** 1.54 § 0.21
2.49 § 0.21* 2.55 § 0.18*
The Real-Time RT-PCR was used to determine the expression of miRNA155 and Apaf-1 in A549 cells treatment with 5 mg mL¡1 Aroclor 1254 for 1, 4 and 24 h. b-actin was used as internal reference. All experiments were repeated at least three times, and the results were presented as mean § standard deviation (s.d.) of three separate determinations. *P < 0.05, **P < 0.01 compared with the control group.
PCR results (Table 1) showed that expression of Apaf1 reached its peak at 4 h, and then gradually reduced. In addition, expression of miRNA155 gradually
Fig. 4. The expression level of Bax, Apaf-1 and Cyt-c in A549 cells after Aroclor 1254 treatment. (A): The expression level of Bax and Apaf-1 in A549 cells after Aroclor 1254 treatment. A549 cells were treated with different concentrations (0, 1, 5 and 10 mg mL¡1) of Aroclor 1254 for 1, 4 or 24 h. The total protein samples were collected and Western blot assay were conducted to evaluate the expression level of related proteins. (B): The expression level of Cyt-c in A549 cells after Aroclor 1254 treatment. A549 cells were treated with different concentrations (0, 1, 5 and 10 mg mL¡1) of Aroclor 1254 for 1, 4 or 24 h. The cytoplasmic protein samples were collected and Western blot assay were conducted to evaluate the expression level of related proteins. GAPDH was used as an internal reference.
Aroclor 1254 induces apoptosis of human A549 lung cancer cells increased with the treatment time, miRNA155 level of Aroclor 1254 group at 24 h was significantly higher than that at 4 h (P < 0.05).
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Discussion Polychlorinated biphenyls (PCBs) were chlorinated biphenyl compounds mainly manufactured between 1930s and 1970s, and PCBs production was gradually ceased in the late 1970s. However, PCBs products are still in use in some older electrical capacitors and transformers, and the release of PCBs into the environment posed potential threats to human health. Workers maintaining or disassembling PCB-containing products are thought to have the highest risk for occupational PCBs exposure. In addition, due to the persistence of PCBs in environment, vaporization of PCBs from landfills and contaminated surface water become a universal exposure route for outdoor and indoor air contamination.[28–30] For those persons who live or work in certain PCBs-contaminated circumstances, inhalation route is a predominated way of PCBs exposure. Although toxicology information of PCBs exposure from digestion source have been carefully assessed and well documented, PCBs exposure from air source is nearly completely ignored until a decade ago. The toxicological importance of atmospheric PCBs has recently become a substantial concern because of their obvious adverse health effects. Pavuk et al.[31] have reported that high level of environmental PCBs exposure in the Michalovce district was associated with occurrence of certain cancers such as stomach and lung cancer. However, the information about the potential molecular mechanism of airborne PCBs toxicity is still deficient. Aroclor 1254 are representative technical product of PCBs mixture, showing high similarity with PCB contamination of e-waste dump site soil. The present study was conducted to investigate the cellular toxicity and potential molecular mechanism of Aroclor 1254 on A549 cells. Aroclor 1254 showed obvious toxicity on A549 cells as evidenced by our findings that Aroclor 1254 inhibited viability of A549 cells and induced cell apoptosis in a concentration-dependent manner. Previous works have shown that PCBs and their metabolites promoted the generation of oxygen reactive compounds that can induce oxidative DNA damage.[32] In this study, the intracellular ROS level in A549 cells treated with different concentration of Aroclor 1254 (1, 5, 10 mg mL¡1) was measured by DCFH-DA assay. ROS in Aroclor 1254-treated groups (for 1 h or 4 h) was significantly elevated as compared with the control group cells. When Aroclor 1254 exposure time prolonged to 24 h, however, elevation of ROS induced by Aroclor 1254 was attenuated or almost recovered. Considering the influence of Aroclor 1254 treatment (for 24 h) on cell viability and
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apoptosis, the attenuation of ROS production may due to dramatically cell death induced by Aroclor 1254. Electron and proton transport through the inner membrane of mitochondria is responsible for cellular ROS generation, and mitochondria-mediated abnormal ROS is widely recognized as an aggravating factor in numerous human diseases.[33] Meanwhile, mitochondrion is a particularly susceptible target of oxidative damage induced by ROS, which could disrupt membrane structure and certain mitochondrial proteins such as succinate dehydrogenase and cytochrome oxidase. Further, mitochondria play an important role in intracellular Ca2C homeostasis and Ca2C mediated signaling under normal physiological conditions.[34] It is reported that a decreased mitochondrial proton gradient and increased mitochondrial Ca2C could synergistically regulate stochastic mitochondrial permeability transition pore (MPTP) opening and bursting mode of ROS production in many types of cells. Ca2C overload in mitochondria serves as a key initiator of cell death in many cell types.[35, 36] Several research groups have reported that PCBs could alter intracellular calcium homeostasis by impairing mitochondrial membrane integrity.[37, 38] A loss of mitochondrial membrane potential (DC) is known to be one of the earliest events in apoptosis, which will induce the opening of the mitochondrial permeability transition pore. In this study, we also found that Aroclor 1254 could induce reduction of mitochondrial membrane potential in a concentration-dependent manner accompanying with elevation of cellular Ca2C level, indicating a loss of plasma membrane integrity and MPTP opening. MPTP opening could also result in release of cytochrome C and AIF mitochondrial apoptotic factor, which consequently lead to cell apoptosis. BCL-2 associated X protein (Bax) is an important factor to mediate MPTP opening, serving as an essential gateway to mitochondrial dysfunction and activation of intrinsic apoptotic pathway.[39] Western blotting results of this study indicated that Aroclor 1254 promoted release of Cyt-c from mitochondria to cytoplasm and increased expression of Bax in a concentration-dependent manner. But after 24 h PCBs exposure, expression of Cyt-c and Bax virtually decreased, which may due to the massive cell death caused by Aroclor 1254 exposure. The miRNA155 plays a role in development and progression of lung cancer. In our previous study, we have proved that apoptotic protease activating factor 1 (Apaf-1) is a target of miRNA155.[40] In this study, we also found that PCBs treatment induced apoptosis in A549 cells and expression of Apaf-1 was negatively regulated by miRNA155. Taken together, Aroclor 1254 had obvious toxic effects in A549 cells as evidenced by suppression of cell viability and increase of cell apoptosis, which may be associated with elevated cellular Ca2C level and ROS production through mitochondrial pathway. Activation of Apaf-1 apoptotic pathway and alteration of Cyt-c, Apaf-1 and
812 miRNA155 expression were involved in Aroclor 1254 induced apoptosis.
Funding The study was supported by grants from the National Natural Science Funds for Distinguished Young Scholars (41225013), the State Key Program of National Natural Science Foundation of China (41130752), the National Science Foundation of China (81072335); Innovative Research Team in University (IRT13078); and the Earmarked Fund of the State Key Laboratory of Organic Geochemistry (DGL-201212).
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Yufan et al. sport-caught fish consumption. Environ Res. 2006, 100, 173–183. [13] Fitzgerald, E.F.; Hwang, S.A.; Gomez, M.; Bush, B.; Yang, B.Z.; Tarbell, A. Environmental and occupational exposures and serum PCB concentrations and patterns among Mohawk men at Akwesasne. J. Expo. Sci. Environ. Epi. 2007, 17, 269–278. [14] Rudel, R.A.; Camann, D.E.; Spengler, J.D.; Korn, L.R.; Brody, J.G. Phthalates, alkylphenols, pesticides, polybrominated diphenyl ethers, and other endocrine-disrupting compounds in indoor air and dust. Environ. Sci. Technol, 2003, 37, 4543–4553. [15] Tue, N.M.; Takahashi, S.; Suzuki, G.; Isobe, T.; Viet, P.H.; Kobara, Y.; Seike, N.; Zhang, G.; Sudaryanto, A.; Tanabe, S. Contamination of indoor dust and air by polychlorinated biphenyls and brominated flame retardants and relevance of non-dietary exposure in Vietnamese informal e-waste recycling sites. Environ. Int. 2013, 51, 160–167. [16] Hu, X.; Adamcakova-Dodd, A.; Lehmler, H.J.; Hu, D.; Hornbuckle, K.; Thorne, P.S. Subchronic inhalation exposure study of an airborne polychlorinated biphenyl mixture resembling the Chicago ambient air congener profile. Environ. Sci. Technol. 2012, 46, 9653–9662. [17] Dallaire, F.; Dewailly, E.; Vezina, C.; Muckle, G.; Weber, J.P.; Bruneau, S.; Ayotte, P. Effect of prenatal exposure to polychlorinated biphenyls on incidence of acute respiratory infections in preschool Inuit children. Environ. Health Perspect. 2006, 11, 1301– 1305. [18] Anderson, L.M.; Ward, J.M.; Fox, S.D.; Isaaq, H.J.; Riggs, C.W. Effects of a single dose of polychlorinated biphenyls to infant mice on N-nitrosodimethylamine-initiated lung and liver tumors. Int. J. Cancer 1986, 38, 109–116. [19] Anderson, L.M.; Logsdon, D.; Ruskie, S.; Fox, S.D.; Issaq, H.J.; Kovatch, R.M.; Riggs, C.M. Promotion by polychlorinated biphenyls of lung and liver tumors in mice. Carcinogenesis 1994, 15, 2245–2248. [20] Foreman, J.; Demidchik, V.; Bothwell, J.H.; Mylona, P.; Miedema, H.; Torres, M.A.; Linstead, P.; Costa, S.; Brownlee C.; Jones, J.D., Davies; J.M.; Dolan, L. Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 2003, 422, 442–446. [21] Ishikawa, K.; Takenaga, K.; Akimoto, M.; Koshikawa, N.; Yamaguchi, A.; Imanishi, H.; Nakada, K.; Honma, Y.; Hayashi, J. ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis. Science 2008, 320, 661–664. [22] Tsukagoshi, H.; Busch, W.; Benfey, P.N. Transcriptional regulation of ROS controls transition from proliferation to differentiation in the root. Cell 2010, 143, 606–616. [23] Xia, C.; Meng, Q.; Liu, L.Z.; Rojanasakul, Y.; Wang, X.R.; Jiang, B.H. Reactive oxygen species regulate angiogenesis and tumor growth through vascular endothelial growth factor. Cancer Res. 2007, 67, 10823–10830. [24] Valavanidis, A.; Vlahogianni, T.; Dassenakis, M.; Scoullos, M. Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants. Ecotoxicol. Environ. Saf. 2006, 64, 178–189. [25] Oakley, G.G.; Devanaboyina, U.; Robertson, L.W.; Gupta, R.C. Oxidative DNA damage induced by activation of polychlorinated biphenyls (PCBs): implications for PCB-induced oxidative stress in breast cancer. Chem Res Toxicol. 1996, 9, 1285–1292. [26] Srinivasan, A.L.; Lehmler, H.J.; Robertson, L.W.; Ludewig, G. Production of DNA strand breaks in vitro and reactive oxygen species in vitro and in HL-60 cells by PCB metabolites. Toxicol Sci. 2001, 60, 92–102. [27] Lee, J.Y.; Kim, J.W.; Lim, H.S.; Joo, W.H.; Cho, Y.K.; Moon, J. Y. Changes in antioxidant defense systems by 2, 20, 5, 50-tetrachlorobiphenyl exposure in neuronal SK-N-MC cells. Toxicol. Lett. 2005, 157, 139–149.
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Aroclor 1254 induces apoptosis of human A549 lung cancer cells [28] Liebl, B.; Schettgen, T.; Kerscher, G.; Broding, H.C.; Otto, A.; Angerer, J.; Drexler, H. Evidence for increased internal exposure to lower chlorinated polychlorinated biphenyls (PCB) in pupils attending a contaminated school. Int. J. Hyg. Environ. Health 2004, 207, 315–324. [29] Persson, N.J.; Pettersen, H.; Ishaq, R.; Axelman, J.; Bandh, C.; Broman, D.; Zebuhr, Y.; Hammar, T. Polychlorinated biphenyls in polysulfide sealants-occurrence and emission from a landfill station. Environ. Pollut. 2005, 138, 18–27. [30] Swackhamer, D.L.; Armstrong, D.E. Estimation of the atmospheric and nonatmospheric contributions and losses of polychlorinated biphenyls for Lake Michigan on the basis of sediment records of remote lakes. Environ. Sci. Technol. 1986, 20, 879–883. [31] Pavuk, M.; Cerhan, J.R.; Lynch, C.F.; Schecter, A.; Petrik, J.; Chovancova, J.; Kocan, A. Environmental exposure to PCBs and cancer incidence in eastern Slovakia. Chemosphere 2004, 54, 1509– 1520. [32] Miller-Perez, C.; Sanchez-Islas, E.; Mucio-Ramirez, S.; MendozaSotelo, J, and Leon-Olea, M. The polychlorinated biphenyls (PCBs) environmental pollutants and their effects on the nervous system and health. Salud Mental. 2009, 32, 335–346. [33] Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M.T.D.; Mazur, M.; Telser, J. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell. Biol. 2007, 39, 44–84.
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[34] Nicholls, D.G. Mitochondria and calcium signaling. Cell Calcium. 2005, 38, 311–317. [35] Hansson, M.J.; Mansson, R.; Morota, S.; Uchino, H.; Kallur, T.; Sumi, T., Ishii, N., Shimazu, M.; Keep, M.F.; Jegorov, A.; Elmer, E. Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition. Free Radic. Biol. Med. 2008, 45, 284–294. [36] Hou, T.; Zhang, X.; Xu, J.; Jian, C.; Huang, Z.; Ye, T.; Hu, K.; Zheng, M.; Gao, F.; Wang, X.; Cheng, H. Synergistic triggering of superoxide flashes by mitochondrial Ca2C uniport and basal reactive oxygen species elevation. J. Biol. Chem. 2013, 288, 4602–4612. [37] Langeveld, W.T.; Meijer, M.; Westerink, R.H. Differential effects of 20 non-dioxin-like PCBs on basal and depolarization-evoked intracellular calcium levels in PC12 cells. Toxicol. Sci. 2012, 126, 487–496. [38] Londo~ no, M; Shimokawa, N; Miyazaki, W.; Iwasaki, T.; Koibuchi, N. Hydroxylated PCB induces Ca2C oscillations and alterations of membrane potential in cultured cortical cells. J. Appl. Toxicol. 2010, 30, 334–342. [39] Hollville, E.; Martin, S.J. Greasing the path to BAX/BAK activation. Cell 2012, 148, 845–846. [40] Zang, Y.S.; Zhong, Y.F.; Fang, Z.; Li, B.; An, J. MiRNA155 inhibits the sensitivity of lung cancer cells to cisplatin via negative regulation of Apaf-1 expression. Cancer Gene Ther. 2012, 19, 773–778.
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Aroclor 1254 inhibits cell viability and induces apoptosis of human A549 lung cancer cells by 2+
modulating the intracellular Ca level and ROS production through the mitochondrial pathway a
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Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai, P. R. China Published online: 01 Jun 2015.
To cite this article: Yufan Zhong, Panpan Guo, Xiu Wang & Jing An (2015) Aroclor 1254 inhibits cell viability and induces 2+
apoptosis of human A549 lung cancer cells by modulating the intracellular Ca level and ROS production through the mitochondrial pathway, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 50:8, 806-813, DOI: 10.1080/10934529.2015.1019797 To link to this article: http://dx.doi.org/10.1080/10934529.2015.1019797
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Journal of Environmental Science and Health, Part A (2015) 50, 806–813 Copyright © Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2015.1019797
Aroclor 1254 inhibits cell viability and induces apoptosis of human A549 lung cancer cells by modulating the intracellular Ca2C level and ROS production through the mitochondrial pathway YUFANG ZHONG, PANPAN GUO, XIU WANG and JING AN
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Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai, P. R. China
To study the acute toxic effects of PCBs on airway exposure, the cell viability, apoptosis and mitochondrial functions of human lung cancer cell line A549 were measured and compared after Aroclor 1254 exposure for different time. The results showed that Aroclor 1254 could inhibit cell viability and increase cell apoptosis in a concentration- and time-dependent manner. The mitochondrial apoptosis pathway was confirmed playing an important role. ROS elevation was an early response within 1h treatment of Aroclor 1254. Then after 4 h of Aroclor 1254 exposure, the intracellular calcium level increased and mitochondrial transmembrane potential (DCm) collapsed, accompanying with Cytochrome c (Cyt-c) leakage, boosting expression of Bax, Apaf-1 and miRNA155, which were involved in the mitochondrial apoptosis pathway. After 24 h of Aroclor 1254 exposure, ROS returned to normal level, but cell apoptosis rate was higher than that at 4 h with DCm continued collapsing and intracellular calcium increased. In conclusion, Aroclor 1254 could suppress cell viability and induce apoptosis in A549 cells, which was associated with ROS over-production and elevated cellular Ca2C level, which may result in mitochondrial dysfunction, inducing expression of Bax/Cyt-c/Apaf-1 and miRNA155. Keywords: Aroclor 1254, A549, ROS, cell apoptosis, mitochondrial membrane potential, cellular Ca2C.
Introduction Polychlorinated biphenyls (PCBs) refer to a family of synthetic organic chemicals known as chlorinated hydrocarbons. PCBs had been widely used in electrical equipment, heat transfer, hydraulic equipment, and many other industrial applications as insulating additives before their ban in 1970s. However, PCBs continue to be released from the PCB-containing consumer products due to improper disposal, and enter environmental media including air, water, and soil. Due to their long half-lives, extreme persistence in environment and bio-accumulation capacity, PCBs exposure are thought to be a significant health hazard for human populations. Chronic exposure to PCBs and their metabolites have multifaceted adverse effects on wild organisms such as developmental deformities,[1,2] immunological defects,[3]
Address correspondence to Jing An, Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, P. R. China; E-mail: [email protected] Received October 27, 2014.
endocrine disruption[4,5] and potential carcinogenesis.[6,7] Results of animal studies and epidemiological studies showed that PCBs could cause cancer development and enhance metastatic propensity in various target organs including liver, brain, breast, and prostate.[8–10] PCBs can be bioaccumulated in the food web, and digestion of contaminated animal foods is regarded as a primary exposure route of PCBs.[11, 12] Some evidences have shown that inhalation uptake is also a significant source route of PCBs exposure.[13,14] Results of daily intake estimation for e-waste recycling workers indicate that air inhalation contributes a large portion of low-chlorinated PCB intake (>80% for triCBs).[15] In urban areas, inhalation of indoor dust and air has been highlighted as an important pathway of PCBs exposure.[16] Recent epidemiologic study found that children living near a hazardous PCBs waste site had higher risks of respiratory diseases.[17] Moreover, several PCB mixtures and congeners were reported to promote lung tumors in a mice model.[18,19] Therefore, assessment of health risk and toxicity mechanisms associated with exposure of airborne PCBs is necessary.
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Aroclor 1254 induces apoptosis of human A549 lung cancer cells Reactive oxygen species (ROS) are well-established inducers of oxidative damage responsible for various biological processes, such as cell viability, proliferation and cell death.[20-23] In the biological systems, ROS are usually used to assess toxic effects resulting from exposure to chemical toxins.[24] Considerable evidence showed that PCBs exposure could cause uncontrolled ROS accumulation and intracellular oxidative stress,[25,26] and eventually lead to various adverse disorders including respiratory system diseases.[27] Aroclor 1254 is a widely used commercial PCBs mixture containing dioxin-like and non-dioxin-like PCBs, as well as several highly potent polychlorinated dibenzo-p-dioxin and -furan impurities. Mitochondrion is susceptive target for chemicals exposure and a hinge of various cytotoxic progresses such as apoptosis. Bax, Cyt-c and Apaf-1 play important role in the mitochondria involved apoptosis. In this study, the toxic effects of Aroclor 1254 in the human lung carcinoma cell line (A549) were evaluated through measurement of cell viability, apoptosis, ROS production, intracellular Ca2C Level, mitochondrial membrane potential (DCm) and Cyt-c release. In addition, expression of miRNA155, Apaf-1 and Bax in A549 cells exposed to Aroclor 1254 was determined to explore the underlying molecular mechanism.
Materials and methods Materials A549 cell line was from American Type Culture Collection (ATCC; Manassas, VA, USA). The Ham’s F-12 culture media and fetal bovine serum (FBS) were purchased from GE (Piscataway, NJ, USA). Aroclor 1254 (CAS no: 11097-69-1) was purchased from AccuStandard (New Haven, CT, USA). Solutions of Aroclor 1254 at strength of 20 mg/mL were prepared in DMSO and diluted as necessary with the cell culture medium. Other chemicals and kits were purchased from the companies as following: Cell Counting Kit-8 (CCK-8), Fluo-4 AM and F-127 from Dojindo (Kumamoto, Japan); Nuclear and Cytoplasmic Extraction Reagent Kit (NE-PER), Mammalian Protein Extraction Reagent (M-PER) and BCA kit from Thermo Fisher Scientific Inc (Waltham, MA, USA); Apoptosis detection kit from Mbchem (Shanghai, China); 20 ,70 dichlorofluorescein diacetate (DCFH-DA), tert-Butyl hydroperoxide (tBHP) and PVDF membrane from Millipore (Darmstadt, Germany); TRIzol from Invitrogen (Paisley, UK); SYBR Green qPCR Master Mix Kit and Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase from TOYOBO (Osaka, Japan); Rabbit monoclonal antibodies for Cytochrome C (Cyt-c), Apaf-1 and Bax from Epitomics (Burlingame, CA, USA), mouse polyclonal antibody for GAPDH from Multisciences Biotechnology (Hangzhou, China); anti-Rabbit IgG(HCL)/HRP
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and anti-Mouse IgG (HCL)/HRP from Dingguo Biotechnology (Beijing, China); Rhodamine 123, DMSO and other analytical grade reagents from Sigma (St. Louis, MO, USA). Absorbance was recorded using iMark plate reader (BioRad, Hercules, CA, USA). Apoptosis was assayed by flow cytometry (Becton Dickinson, San Jose, CA, USA) and data was analyzed using Cell Quest Software (Becton Dickinson, San Jose, CA, USA). Fluorescence was detected by fluorescence microscope (Olympus BX-51, Tokyo, Japan) and analyzed by Image-pro Plus 6.0 software (IPP, Media Cybernetics, Rockville, MD, USA). Quantitative PCR ran in ABI Prism 7500 Sequence Detection system (Applied Biosystems, Waltham, MA, USA). The optical densities of western blotting bands were quantized using the Chemi-Imager digital imaging system (Alpha Innotech, San Leandro, CA, USA). Cell culture and treatments A549 cells was cultured in 10% complete medium (Ham’s F-12 medium, 10% fetal bovine serum, 2 mM glutamine, 0.33% sodium bicarbonate, 100 units mL¡1 penicillin and 0.1 mg/mL streptomycin) in humidified incubator with 5% CO2 at 37℃. Before Aroclor 1254 treatment, A549 cells at exponentially growing phase were seeded in culture plates and grown for 24 h to 70–80% confluence. Aroclor 1254 were dissolved in DMSO and diluted with culture media to 0, 1, 5 and 10 mg/mL with a final concentration of DMSO at 0.1% v/v. After Aroclor 1254 treatment (1 h, 4 h or 24 h), cells were washed with D-Hanks and subjected to following assay procedures. All experiments were carried out in at least triplicate, and 3 parallel samples were tested for each concentration (6 parallel samples were applied in cck-8 test). Cell viability First, 5 £ 103 cells well¡1 seeded in 96-well plates were treated with different concentrations of Aroclor 1254 (0, 1, 5, 10 mg mL¡1) for 24 h, and cell viability was measured by Cell Counting kit-8 (CCK-8) as the description of kit. In brief, 10 mL of CCK-8 reagent in 90 mL Ham’s F-12 medium per well was added to cell culture medium, and then incubated at 37℃ for 1 h. Absorbance of each well was measured at 450 nm with an iMark plate reader. Assessment of apoptosis Apoptosis of A549 cells was measured using flow cytometry with Annexin V-FITC/PI double staining assay kit. After treatment with various concentrations of Aroclor 1254, cells were dispersed with 0.25% trypsin, then washed twice with D-Hank’s and suspended at a concentration of 1 £ 106 cells mL¡1 by 1 £ binding buffer (10 mM
808 HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). After 30 min incubation at 37℃ in darkness with Annexin V-FITC and propidium iodide (PI), cells were immediately analyzed by flow cytometry using the FL2 detector at 488 nm excitation wavelength.
Yufan et al. amounts of protein (60 mg per lane) were subjected to 10% SDS-PAGE separation and transferred (semidry transfer assay) from the gel onto an ethanol saturated PVDF membrane, which was then blocked by BSA and incubated with primary antibodies overnight at 4℃, blotted with secondary antibodies for 1 h at room temperature. Finally blots were visualized using chemiluminescence assay.
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Measurement of reactive oxygen species (ROS) When combined with the hydrogen peroxide, ROS probe DCFH-DA will convert to DCF, which emits bright green fluorescence. To evaluate the intracellular ROS generation induced by Aroclor 1254, 1 £ 106 cells per dish seeded in 35-mm culture dishes were treated with Aroclor 1254, then cells were washed twice with D-Hanks and loaded with 10 mM DCFH-DA at 37℃ for 30 min in darkness. Finally, cells were washed three times again with D-Hanks, and DCF fluorescence was measured by fluorescence microscope. Measurement of mitochondrial membrane potential (DCm) Rhodamine 123 (Rh123) is a membrane permeable fluorescent cationic dye, which is selectively taken up by mitochondria proportional to the Mitochondrial membrane potential. A549 cells cultured in 35-mm culture dishes were treated with different concentrations of Aroclor 1254 (0, 1, 5, 10 mg mL¡1) for 1, 4 or 24 h, then cells were washed twice with D-Hanks and incubated with 10 mg mL¡1 Rh123 at 37℃ for 30 min in darkness. Finally, cells were washed three times and fluorescence intensity was measured under fluorescence microscope. Measurement of intracellular Ca2C level Intracellular calcium levels were determined by ratiometric fluorimetry with the fluorescent calcium indicator Fluo-4 AM. After Aroclor 1254 treatments, approximately 1 £ 106 of A549 cells were seeded on 35-mm culture dishes, and rinsed 3 times with D-Hanks. Then cells were mixed with 4 mM Fluo-4 AM in the buffer for 30 min at 37℃, followed with washing three times with D-Hanks. Calcium imaging was conducted with fluorescence microscope at 488 nm wavelength. In this process, F-127 (0.05%, W/V) was used to prevent Fluo-4 AM from polymerization in the D-Hank’s solution. Western blotting For every sample, 2 £ 107 cells were lysed using the NEPER Kit or M-PER Kit to obtain cytoplasmic extracts or cell total protein respectively. The cytoplasmic extracts were used to measure the leakage of Cyt-c, and cell total protein was used to measure the expression of Apaf-1 and Bax. GAPDH was used as the internal reference. Equal
Real-time PCR Total RNA of A549 cells was extracted using TRIzol (1 £ 106 cells mL¡1), then reversely transcribed to cDNA using M-MLV reverse transcriptase. Approximately 1 mg cDNA for each sample was amplified in a 20-mL reaction mixture, which contains SYBR Green PCR Master Mix, appropriate primer pairs and nuclease-free water. The qPCR cycling conditions were as follow: 40 cycles of 94℃ for 10 s, 59℃ for 20 s, and 72℃ for 10 s. All samples were measured in triplicate with the human housekeeping gene b-actin being used as a reference to normalize data. The qPCR primer sets were listed as follows: miRNA155, 50 CTCTAATGGTGGCACAAA-30 (forward), and 50 TGATAAAAACAAACATGGGCTTGAC-30 (reverse); Apaf-1, 50 -GGAGGAC- CCTCAAGAGGATATG-30 (forward) and 50 -GGATTTCTCCCAATAG- GCCACT30 (reverse); b-actin, 50 -CCATGGAGAAGGCTGGGG-30 (forward) and 50 -CAAAGTGTCATG- GATGACC-30 (reverse). Statistical analyses All experiments were conducted in at least triplicate repeated wells for each sample, and data were presented as mean § standard deviation derived from three independent experiments. Statistical differences were conducted using SPSS 11.0 software (SPSS Inc., Chicago, IL, USA). The differences between control and exposed groups were evaluated by ANOVA. P values < 0.05 were considered to be statistically significant.
Results Aroclor 1254 could inhibit cell viability and increase apoptosis of A549 cells A549 cells were exposed to different concentrations of Aroclor 1254 and CCK-8 assay was conducted to determine the cell viability. As shown in Figure 1A, after Aroclor 1254 treatment, the cell viability obviously decreased in a concentration- and time-dependent manner (P < 0.05). Compared with the control group, the cell viability of 10 mg mL¡1 Aroclor 1254 groups reduced to 78% after 4 h treatment, 5 and 10 mg/mL groups significantly reduced to 49% and 24% after
Aroclor 1254 induces apoptosis of human A549 lung cancer cells
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Aroclor 1254 increased ROS production Intracellular ROS production was measured by DCFHDA assay. As shown in Figure 2, after Aroclor 1254 treatment for 1 h, the DCFH-DA fluorescence density of A549 cells was significantly (P < 0.01) elevated as compared with the control cells in a dose-dependent manner. After treatment for 4 h, Aroclor 1254 induced ROS elevation in all three treatment groups, while the increasing trend slowed (P < 0.05); and after 24 h treatment, the ROS level difference between control group and treatment groups disappeared (P > 0.05).
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Effects of Aroclor 1254 on mitochondrial membrane potential and intracellular Ca2C level
Fig. 1. The cell survival and apoptosis of A549 after treatment with Aroclor 1254 (A): The cell survival of A549 after treatment with different concentration (0, 1, 5 and 10 mg mL¡1) of Aroclor 1254 for1, 4, 24 h. CCK8 assay was used to evaluate the cell survival. (B): The apoptosis of A549 after treatment with different concentration (0, 1, 5 and 10 mg mL¡1) of Aroclor 1254 for1, 4, 24 h. The cell apoptosis was assessed by Annexin V-FITC/PI double staining assay. *P < 0.05, **P < 0.01 compared with the control group.
The Dcm was measured with Rhodamine 123 (Rh123) assay results (Fig. 3A) showed that Aroclor 1254 treatment induced loss of Dcm in a dose-dependent manner. In groups treated with 10 mg mL¡1 of Aroclor 1254, the Dcm significantly decreased compared with that of control group at 4 h or 24 h (P < 0.05); for 5 mg mL¡1 group, the Dcm loss appeared only at 24 h. In the 1 mg mL¡1 group,
24 h, respectively. Annexin V-FITC/PI double staining assay results (Fig. 1B) showed that Aroclor 1254 induced cell apoptosis in a time- and concentrationdependent manner. After Aroclor 1254 treatment, the apoptotic cell percentage of 5 and 10 mg mL¡1 groups was significantly higher than that of the control group; and the apoptosis rate at 24 h were even higher than that at 4 h both in 5 and 10 mg mL¡1 groups (Fig. 1).
Fig. 2. The ROS production induced by Aroclor 1254 treatment. A549 cells were treated with Aroclor 1254 (0, 1, 5 and 10 mg mL¡1) for 1, 4 or 24 h. Then the intracellular ROS level measured with DCFH-DA assay. *P < 0.05, **P < 0.01 compared with the control group.
Fig. 3. The Mitochondrial Membrane Potential and cellular Ca2C level of A549 after treatment with Aroclor 1254: (A): The Mitochondrial Membrane Potential of A549 after treatment of different concentration (0, 1, 5 and 10 mg mL¡1) of Aroclor 1254 for 1, 4 or 24 h. Rhodamine 123 (Rh123) assay was used to evaluate the Mitochondrial Membrane Potential. (B): The cellular Ca2C Level of A549 after treatment of different concentration (0, 1, 5 and 10 mg mL¡1) of Aroclor 1254 for 1, 4 or 24 h. For calcium measurement, cells after Aroclor 1254 treatment were incubated with Fluo 4-AM. Fluorescence images were recorded with a fluorescence microscope. *P < 0.05, **P < 0.01 compared with the control group.
810 no obvious Dcm decrease was observed. Measurement of Ca2C level was conducted with Fluo-4 AM/F-127 assay and the intensity of fluorescence was analyzed by Image-pro plus 6.0 software. Figure 3B showed that 5 and 10 mg mL¡1 Aroclor 1254 induced significant elevation of Ca2C level at 4 to 24 h (P < 0.05), while 1 mg mL¡1 group caused Ca2C level elevation only at 24 h (P < 0.05).
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Expression of apoptosis-related proteins induced by Aroclor 1254 Western blotting results showed that expression level of Bax and Apaf-1 (Fig. 4A) increased after Aroclor 1254 treatment, which peaked at 4 h, and recovered after 24 h treatment. In addition, the cytoplasmic Cyt-c (Fig. 4B) also increased after Aroclor 1254 treatment for 1 h, reached peak at 4 h, and then attenuated. Consistent with the Western blotting results, the real-time
Yufan et al. Table 1. The expression level of Apaf-1 and miRNA155 in A549 cells after Aroclor 1254 treatment. Groups Apaf-1 miRNA155
1h
4h
24 h
1.05 § 0.06 1.45 § 0.15
3.23 § 0.32** 1.54 § 0.21
2.49 § 0.21* 2.55 § 0.18*
The Real-Time RT-PCR was used to determine the expression of miRNA155 and Apaf-1 in A549 cells treatment with 5 mg mL¡1 Aroclor 1254 for 1, 4 and 24 h. b-actin was used as internal reference. All experiments were repeated at least three times, and the results were presented as mean § standard deviation (s.d.) of three separate determinations. *P < 0.05, **P < 0.01 compared with the control group.
PCR results (Table 1) showed that expression of Apaf1 reached its peak at 4 h, and then gradually reduced. In addition, expression of miRNA155 gradually
Fig. 4. The expression level of Bax, Apaf-1 and Cyt-c in A549 cells after Aroclor 1254 treatment. (A): The expression level of Bax and Apaf-1 in A549 cells after Aroclor 1254 treatment. A549 cells were treated with different concentrations (0, 1, 5 and 10 mg mL¡1) of Aroclor 1254 for 1, 4 or 24 h. The total protein samples were collected and Western blot assay were conducted to evaluate the expression level of related proteins. (B): The expression level of Cyt-c in A549 cells after Aroclor 1254 treatment. A549 cells were treated with different concentrations (0, 1, 5 and 10 mg mL¡1) of Aroclor 1254 for 1, 4 or 24 h. The cytoplasmic protein samples were collected and Western blot assay were conducted to evaluate the expression level of related proteins. GAPDH was used as an internal reference.
Aroclor 1254 induces apoptosis of human A549 lung cancer cells increased with the treatment time, miRNA155 level of Aroclor 1254 group at 24 h was significantly higher than that at 4 h (P < 0.05).
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Discussion Polychlorinated biphenyls (PCBs) were chlorinated biphenyl compounds mainly manufactured between 1930s and 1970s, and PCBs production was gradually ceased in the late 1970s. However, PCBs products are still in use in some older electrical capacitors and transformers, and the release of PCBs into the environment posed potential threats to human health. Workers maintaining or disassembling PCB-containing products are thought to have the highest risk for occupational PCBs exposure. In addition, due to the persistence of PCBs in environment, vaporization of PCBs from landfills and contaminated surface water become a universal exposure route for outdoor and indoor air contamination.[28–30] For those persons who live or work in certain PCBs-contaminated circumstances, inhalation route is a predominated way of PCBs exposure. Although toxicology information of PCBs exposure from digestion source have been carefully assessed and well documented, PCBs exposure from air source is nearly completely ignored until a decade ago. The toxicological importance of atmospheric PCBs has recently become a substantial concern because of their obvious adverse health effects. Pavuk et al.[31] have reported that high level of environmental PCBs exposure in the Michalovce district was associated with occurrence of certain cancers such as stomach and lung cancer. However, the information about the potential molecular mechanism of airborne PCBs toxicity is still deficient. Aroclor 1254 are representative technical product of PCBs mixture, showing high similarity with PCB contamination of e-waste dump site soil. The present study was conducted to investigate the cellular toxicity and potential molecular mechanism of Aroclor 1254 on A549 cells. Aroclor 1254 showed obvious toxicity on A549 cells as evidenced by our findings that Aroclor 1254 inhibited viability of A549 cells and induced cell apoptosis in a concentration-dependent manner. Previous works have shown that PCBs and their metabolites promoted the generation of oxygen reactive compounds that can induce oxidative DNA damage.[32] In this study, the intracellular ROS level in A549 cells treated with different concentration of Aroclor 1254 (1, 5, 10 mg mL¡1) was measured by DCFH-DA assay. ROS in Aroclor 1254-treated groups (for 1 h or 4 h) was significantly elevated as compared with the control group cells. When Aroclor 1254 exposure time prolonged to 24 h, however, elevation of ROS induced by Aroclor 1254 was attenuated or almost recovered. Considering the influence of Aroclor 1254 treatment (for 24 h) on cell viability and
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apoptosis, the attenuation of ROS production may due to dramatically cell death induced by Aroclor 1254. Electron and proton transport through the inner membrane of mitochondria is responsible for cellular ROS generation, and mitochondria-mediated abnormal ROS is widely recognized as an aggravating factor in numerous human diseases.[33] Meanwhile, mitochondrion is a particularly susceptible target of oxidative damage induced by ROS, which could disrupt membrane structure and certain mitochondrial proteins such as succinate dehydrogenase and cytochrome oxidase. Further, mitochondria play an important role in intracellular Ca2C homeostasis and Ca2C mediated signaling under normal physiological conditions.[34] It is reported that a decreased mitochondrial proton gradient and increased mitochondrial Ca2C could synergistically regulate stochastic mitochondrial permeability transition pore (MPTP) opening and bursting mode of ROS production in many types of cells. Ca2C overload in mitochondria serves as a key initiator of cell death in many cell types.[35, 36] Several research groups have reported that PCBs could alter intracellular calcium homeostasis by impairing mitochondrial membrane integrity.[37, 38] A loss of mitochondrial membrane potential (DC) is known to be one of the earliest events in apoptosis, which will induce the opening of the mitochondrial permeability transition pore. In this study, we also found that Aroclor 1254 could induce reduction of mitochondrial membrane potential in a concentration-dependent manner accompanying with elevation of cellular Ca2C level, indicating a loss of plasma membrane integrity and MPTP opening. MPTP opening could also result in release of cytochrome C and AIF mitochondrial apoptotic factor, which consequently lead to cell apoptosis. BCL-2 associated X protein (Bax) is an important factor to mediate MPTP opening, serving as an essential gateway to mitochondrial dysfunction and activation of intrinsic apoptotic pathway.[39] Western blotting results of this study indicated that Aroclor 1254 promoted release of Cyt-c from mitochondria to cytoplasm and increased expression of Bax in a concentration-dependent manner. But after 24 h PCBs exposure, expression of Cyt-c and Bax virtually decreased, which may due to the massive cell death caused by Aroclor 1254 exposure. The miRNA155 plays a role in development and progression of lung cancer. In our previous study, we have proved that apoptotic protease activating factor 1 (Apaf-1) is a target of miRNA155.[40] In this study, we also found that PCBs treatment induced apoptosis in A549 cells and expression of Apaf-1 was negatively regulated by miRNA155. Taken together, Aroclor 1254 had obvious toxic effects in A549 cells as evidenced by suppression of cell viability and increase of cell apoptosis, which may be associated with elevated cellular Ca2C level and ROS production through mitochondrial pathway. Activation of Apaf-1 apoptotic pathway and alteration of Cyt-c, Apaf-1 and
812 miRNA155 expression were involved in Aroclor 1254 induced apoptosis.
Funding The study was supported by grants from the National Natural Science Funds for Distinguished Young Scholars (41225013), the State Key Program of National Natural Science Foundation of China (41130752), the National Science Foundation of China (81072335); Innovative Research Team in University (IRT13078); and the Earmarked Fund of the State Key Laboratory of Organic Geochemistry (DGL-201212).
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