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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 65  |  Issue : 4  |  Page : 199-208

Effects of taurine against benzo[α]pyrene-induced cell cycle arrest and reactive oxygen species-mediated nuclear factor-kappa B apoptosis via reduction of mitochondrial stress in A549 cells


1 Department of Nursing, University of Kang Ning, Taipei, Taiwan
2 Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan
3 Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan

Date of Submission23-Mar-2022
Date of Decision17-May-2022
Date of Acceptance08-Jun-2022
Date of Web Publication26-Aug-2022

Correspondence Address:
Prof. Bi-Yu Liu
Department of Nursing, University of Kang Ning, Taipei
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0304-4920.354803

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  Abstract 


Taurine is a free amino acid that prevents reactive oxygen species (ROS) formation. ROS production is associated with oxidative stress, cell proliferation, apoptosis, inflammation, and DNA alterations in benzo[α]pyrene (BaP)-induced lung cells. Here, we assessed the effect of adding of 25 mM taurine on human pulmonary alveolar epithelial A549 cells treated with different concentrations of BaP. After culturing for 24 h, the cells were tested for biomarkers including cell viability, cellular morphology, Annexin V-FITC/propidium iodide, cell cycle regulation, ROS accumulation, mitochondrial membrane potential (MMP), and expression of related signaling genes and proteins. BaP induced cell cycle arrest and decreased cell viability in a dose-dependent manner. In addition, 50 μM BaP induced a 52.2% increase in ROS levels and inhibited MMP by up to 80%; however, taurine decreased BaP-induced ROS production by 19.5% and restored MMP. The expression of nuclear factor-kappa B (NF-κB), B-cell lymphoma-2 (BCL-2) homologous antagonist killer (Bak), BCL-2-associated X protein (Bax), and cytochrome c at both the mRNA and protein levels were increased, and the expression of BCL-2 and BCL-x1 was decreased by BaP treatment. Furthermore, BaP activated caspase-3/7 expression by up to 25%. However, taurine decreased the expression of NF-κB, Bak, Bax and cytochrome c levels, reduced caspase-3/7 activities, and increased the expression of BCL-2 and BCL-x1. Hence, taurine attenuates BaP-induced oxidative stress and mitochondrial dysfunction by inhibiting the NF-κB-mediated intrinsic apoptosis pathway in A549 cells. Taurine can be considered as a preventive molecule to prevent lung damage.

Keywords: Apoptosis, benzo[α]pyrene, mitochondrial stress, oxidative stress, taurine


How to cite this article:
Liu BY, Chiou JZ, Huang KM, Chen TY, Hwang DF. Effects of taurine against benzo[α]pyrene-induced cell cycle arrest and reactive oxygen species-mediated nuclear factor-kappa B apoptosis via reduction of mitochondrial stress in A549 cells. Chin J Physiol 2022;65:199-208

How to cite this URL:
Liu BY, Chiou JZ, Huang KM, Chen TY, Hwang DF. Effects of taurine against benzo[α]pyrene-induced cell cycle arrest and reactive oxygen species-mediated nuclear factor-kappa B apoptosis via reduction of mitochondrial stress in A549 cells. Chin J Physiol [serial online] 2022 [cited 2022 Oct 6];65:199-208. Available from: https://www.cjphysiology.org/text.asp?2022/65/4/199/354803




  Introduction Top


Cancer is a serious disease that is a major health concern globally. Lung cancer is known to be associated with environmental factors, such as air pollution and cigarette smoking in humans. Polycyclic aromatic hydrocarbons, such as benzo[α]pyrene (B[α]P; BaP), an important toxic component of cigarette smoke, induce cell proliferation, apoptosis, inflammation, and DNA alterations, leading to lung cancer.[1],[2],[3],[4], In addition, BaP induces overproduction of reactive oxygen species (ROS), which can damage sensitive biological structures, such as mitochondrial DNA, alter membrane permeability, as well as influence calcium homeostasis and induce endoplasmic reticulum (ER) stress, causing cell apoptosis.[5]

Moreover, apoptosis is activated by caspases (cysteine aspartyl-specific proteases) to change normal cell function. The caspases include initiator (caspase-2, -8, -9, and -10) and executioner (caspase-3, -6, and -7).[6],[7] Upon their activation, executioner caspases can cleave target proteins that eventually lead to cell death.[8] The effective initiator pathway is regulated by B-cell lymphoma-2 (BCL-2) proteins, including pro-apoptotic effector proteins, pro-apoptotic BH3-only proteins, and anti-apoptotic BCL-2 proteins. Activation of BCL-2 proteins can suppress BCL-2-associated X protein (Bax) and BCL-2 homologous antagonist killer (Bak) expression to inhibit cell apoptosis.[9] BH3 only proteins inhibit BCL-2 proteins to induce apoptosis.[8]

Taurine (2-aminoethane sulfonic acid), a non-protein amino acid, is present in a variety of organs in mammals and is present at high concentrations in seafood, especially in squid and mussel (about 5% in dry weight).[10] By contrast, the concentration of taurine in the mature central nervous system and mitochondria of the heart are 25 M and 25 mM, respectively.[11],[12] Another, the concentration of taurine in the human plasma and whole blood was 44 ± 8 and 227 ± 35 mumol/L, respectively.[13] However, the level of taurine in the lung tissue was not report unit now. Various experimental studies have shown that taurine is an essential antioxidant that reduces ROS production and ER stress and mediates recovery of mitochondrial dysfunction caused by heavy metal toxicity, such as aluminum and arsenic insult in neuronal cells.[10],[14] Moreover, taurine can prevent hepatic steatosis, hepatic necrosis, atherosclerotic diseases, hyperlipidemia, and neurotoxicity, as demonstrated in vitro and in vivo studies.[15],[16],[17]

BaP induces oxidative stress and causes lung cancer as shown previously. By contrast, taurine is an antioxidant that prevents ROS insult. It is still unknown whether PaP-induced lung cancer signaling pathway could be inhibited by taurine. In this study, BaP-induced cell apoptosis was used to observe the signaling pathway. The protective mechanism of taurine was also investigated.


  Materials and Methods Top


Chemicals and reagents

DEME medium, DPBS/modifite, and trypsin were purchased from Gibco (Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). Taurine, HEPES, sodium bicarbonate (NaHCO3), SYBR, BaP, 2,7-dichlorofluorescin diacetate (DCFDA), and penicillin–streptomycin sodium were purchased from Sigma (St. Louis, MO, USA). The CytoScanTM WST-1 cell cytotoxicity assay kit was purchased from G-Biosciences (USA). The GeneJET RNA purification kit, caspase-3/7 green detection reagent, Thermo Scientific maximum first-strand cDNA synthesis kit, and primers were purchased from Thermo Fisher (USA). The TUNEL Andy Fluor™ 488 apoptosis detection kit was purchased from Gene Copoeia™ (USA). A quick apoptosis DNA ladder detection kit was purchased from Biovision Biotechnology (Toronto, Ontario, Canada). Rhodamine 123 and propidium iodide (PI) were purchased from Biotium (USA). The Annexin V-FITC assay kit was purchased from Cayman Chemical (USA). Goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG antibodies were purchased from Arigo (USA). Other chemicals used in the study were of cell culture grade and were purchased from common sources.

Preparation of benzo[α]pyrene solutions

A stock solution of BaP was prepared by dissolving it in 99% acetone at a concentration of 50 μM. The working BaP solutions with concentrations of 2, 4, 10, and 50 μM were prepared from the sock solution by diluting in DEME medium.

Cell culture and experimental setup

The human pulmonary alveolar epithelial A549 cell line was obtained from the Department of Food Science at the National Taiwan Ocean University (Keelung, Taiwan). The cell line was incubated with DMEM/F12 (1:1 ratio) medium supplemented with 10% FBS, antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin), 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids in a 75-cm2 flask, and maintained in a humidified incubator with 5% CO2 at 37°C. The experiment included three groups: control, BaP, and BaP + taurine groups. In the control group, the cells were cultured in medium without BaP and taurine supplementation. In the BaP groups, the cells were cultured in medium supplemented with different concentrations of BaP. In the BaP + taurine groups, the cells were cultured in medium supplemented with different concentrations (2–50 μM) of BaP and 25 mM taurine.

Determination of cell viability

Cell viability was determined using the WST-1 assay. A549 cells were dispensed in 96-well plates, and each well contained 1 × 103 cells that were incubated at 37°C in a humidified atmosphere of 5% CO2 for 24 h. The next day, the medium was removed and fresh medium with or without 2, 4, 10, or 50 μM BaP and 25 mM taurine was added and maintained for 24 h. After 24 h of incubation, 100 μl WST-1 was added in each well at 37°C for 4 h. The absorbance was measured at 420–480 nm using an ELISA plate reader. The relative percentage of viable cells was calculated.[18]

% Cytotoxicity = (100× (Control cells – Experimental cells)) ÷ (Control cells)

Determination of cell cycle

The cell cycle was examined using PI staining.[19] A total of 8 × 105 cells were seeded in each well of a 6-well plate. The next day, the medium was removed and fresh medium was added with or without 25 mM taurine and different concentrations (2, 4, 10, and 50 μM) of BaP and further maintained for 24 h. After the incubation period, the cells were fixed in 70% alcohol at − 20°C overnight. Cells were stained with 50 μg/ml PI solution (50 g/ml PI, 0.1 mg/ml RNase A, 0.05% Triton X-100) for 40 min to facilitate selection of the viable cell population at 37°C. After the incubation period, the cell pellet was washed with phosphate buffered saline (PBS) and analyzed on a Becton-Dickinson FACScan flow cytometer, which employed the CellQuest software.

Determination of cell apoptosis

Cells were treated with or without 25 mM taurine and different concentrations (2, 4, 10, and 50 μM) of BaP for 24 h. Cell apoptosis was examined using a double-labeling with Annexin V-FITC (0.025 μg/ml) and PI (5 μl) to discriminate between apoptotic and necrotic cells. The four quadrants included Q1, Q2, Q3 and Q4 were differentiated into necrosis, early apoptosis, live, late apoptosis of cells, respectively, based on identification of the flow cytometry and fluorescence microscopy. Furthermore, another set of cells were subjected to TUNEL staining to detect DNA fragmentation in apoptotic cells. Analysis was performed qualitatively using a Becton-Dickinson FACScan flow cytometer, which employed CellQuest software.[20]

Determination of intracellular reactive oxygen species

Intracellular oxidative stress was estimated as an increase in DCFDA fluorescence using inverted fluorescence microscopy and flow cytometry. DCFDA was converted to fluorescent DCH by peroxides and retained inside the cells.[21] A total of 8 × 105 cells were seeded in a 6-well plate. The next day, the medium was removed and fresh medium was added with or without 25 mM taurine and different concentrations (2, 4, 10, and 50 μM) of BaP, and incubation was continued for additional 24 h. Then, cells were incubated with 10 M DCFDA in serum-free DMEM-F12 medium at 37°C for 30 min. Following staining, the medium was removed and the cells were washed with ice-cold PBS. Subsequently, cells were observed using an inverted fluorescence microscope (Nikon, Japan) and analyzed on a Becton-Dickinson FACScan flow cytometer.

Determination of mitochondrial membrane potential

Mitochondrial membrane potential (MMP) was determined using fluorescent rhodamine 123.[19] Briefly, 8 × 105 cells were incubated with or without BaP and taurine overnight. After 24 h, the medium was removed, and the cells were stained with 10 μM rhodamine 123 at 37°C for 30 min. Then, the cells were harvested with 0.25% trypsin and analyzed on a Becton-Dickinson FACScan flow cytometer.

Quantitative real-time polymerase chain reaction

Gene expression was examined using quantitative polymerase chain reaction (qPCR). Total RNA was extracted using the pure-link™ RNA mini kit. The cells were cultured in DMEM containing 10% FBS cells in a 10 cm dish and incubator at 37°C and 5% CO2 for 24 h. On the next day, the cells treated with fresh medium absence BaP or containing BaP (2, 4, 10, and 50 μM). After incubating for 24 h, the DMEM medium was removed and add trypsin in cells incubator for 5 min, centrifuge 250 g x 5 min. Removes the supernatant (precipitated cells can be directly used RNA Isolation and stored in −70°C) cDNA was synthesized using the HiscripItm first-strand cDNA synthesis kit system for qPCR. The qPCR of Bax, Bak, Bcl-2, and GAPDH genes was carried out using the iQ SYBR Green Supermix system (Bio-Rad, USA). The gene expression results for Bax, Bak, and Bcl-2 were normalized to GAPDH levels. The primer sequences are as following:

Bax-forward 5'-CCTGTGCACCAAGGTGCCGGAACT-3'

Bax-reverse 5'-CCACCCTGGTCTTGGATCCAGCCC-3'

Bak-forward 5'-TTCTGGAAGATCAGCACCCT-3'

Bak-reverse 5'-AGAGTTGAGCAGGACCTTGG-3'

Bcl-2-forward 5'-TTGTGGCCTTCTTTGAGTTCGGTG-3'

Bcl-2-reverse 5'-GGTGCCGGTTCAGGTACTCAGTCA-3'

GAPDH-forward 5'-CCCCTTCATTGACCTCAACTACAT-3'

GAPDH-reverse 5'-CGCTCCTGGAAGATGGTGA-3'.

Western blotting

After the experimental period, cells were lysed in radio-immunoprecipitation assay buffer containing 1% (v/v) protease inhibitor cocktail on ice for 15 min. After centrifugation (20,800 × g) for 10 min, the protein supernatant was collected. The proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Subsequently, the membrane was blocked by placing it in Tris buffer saline (pH 7.5) containing 0.1% tween-20 (TBST) and 5% nonfat milk powder for 2 h at room temperature. Then, the membrane was washed twice with TBST and subsequently incubated with a corresponding primary antibody (1:500) overnight and an HRP-conjugated secondary antibody (1:5000) at room temperature for 2 h. Finally, the membrane was washed twice with TBST and protein bands were visualized using an electrochemiluminescence kit and an imaging system. The intensity of the protein bands was quantified using Quantity One software and considered as the amount of expressed Bak, Bax, cytochrome c, Bcl-2, Bcl-x1, p-nuclear factor-kappa B (p-NF-κB) p65, NF-κB p65, and β-actin. The quantitative results are presented as the mean values of three independent experiments.

Statistical analyses

The results are presented as mean ± standard error of the mean values of three independent experiments and statistically analyzed using analysis of variance followed by Tukey's test. Statistical significance was set at P < 0.05.


  Results Top


Effect of taurine on cell viability by benzo[α]pyrene treatment in A549 cells

As compared to control group, treatment with 0.25 μM BaP resulted in a statistically not significant reduction in cell viability [Figure 1]a. High concentrations of BaP, especially 50 and 100 μM, significantly increased cell death by 90% [Figure 1]a. The dose of 50 μM of BaP was also selected for the subsequent experiments. The concentration of 25 mM of taurine was selected according to the reports of Liu et al. and Chou et al.[10],[14] In BaP + 25 mM taurine group, cell survival rate was maintained at 40%–78%, and the survival rate could be maintained at 40% in the BaP + 25 mM taurine group compared to 23% in 50 μM BaP group [Figure 1]b.
Figure 1: BaP on the viability in A549 cells and the protective effect of taurine. (a) Cells were exposed to different concentrations of BaP for 24 h and cell viability was determined using the WST-1 assay. Results were expressed as percentage of cell viability and expressed as mean ± SEM (N = 3). aP < 0.05 versus untreated BaP group (0 μM, control); bP < 0.05 versus treated with BaP 0.25 μM; cP < 0.05 versus treated with 0.5 μM. (b) Cells were co-treated with or without 25 mM taurine for 24 h and the cell viability was determined using the WST-1 assay. Results are expressed as percentage of cell viability and expressed as mean ± SEM (N = 3). aP < 0.05 versus treated with BaP. BaP: Benzo[α]pyrene, SEM: Standard error of the mean.

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Effect of taurine on cell cycle by benzo[α]pyrene treatment in A549 cells

According to the cell viability experiment, we investigated whether taurine prevented BaP-induced cell cycle arrest in A549 cells. Flow cytometric analysis indicated an increase in the cell population (from 0% to 60%) in the G0/G1 phase and a decrease in the live cell population (from 97% to 24%) after BaP treatment for 24 h [Figure 2]a, [Figure 2]b and [Figure 2]d. However, co-treatment with 25 mM taurine resulted in an increase in the cell population (from 3% to 20%) in G2/M phase and a decrease in the G0/G1 phase (from 71% to 58%) when compared with 50 μM BaP treatment for 24 h [Figure 2]c and [Figure 2]e. These data indicated that taurine prevented cell cycle arrest in A549 cells when used in combination with BaP treatment.
Figure 2: Effect of BaP on cell cycle and the protective effect of taurine in A549 cells. Cells were treated with different concentrations of BaP for 24 h and the number of cells in the various phases of the cell cycle expression was determined using propidium iodide (PI) staining by using a flow cytometer. (a) Distribution of cells in the various phases following BaP treatment. P2 (red fluorescence) indicated total cells; P3 (green fluorescence) indicated dead cells. (b) The percentage of A549 cells in the G0/G1 phase following treatment with 0 μM (untreated control) and BaP (2-50 μM). (c) The percentage of A549 cells in the G0/G1 phase following treatment with 0 μM BaP (untreated control) and BaP (2-50 μM) in combination with 25 mM taurine. (d) Quantification of the data from A–B, which are presented as the mean ± SEM. (e) Quantification of the data from C, which are presented as the mean ± SEM. BaP: Benzo[α]pyrene, SEM: Standard error of the mean.

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Effect of taurine on intracellular reactive oxygen species accumulation after benzo[α]pyrene treatment of A549 cells

DCFHDA fluorescence staining was used to detect the levels of intracellular ROS. BaP treatment for 24 h resulted was increased in green fluorescence [Figure 3]a. However, co-treatment with 25 mM taurine decreased BaP-induced fluorescence [Figure 3]b. The results showed that the intracellular ROS levels were significantly increased at different concentrations (2, 4, 10, and 50 μM) of BaP groups when compared to those in the control group (from 36.0–42.2, 36.0–45.3, 36.0–41.7, and 36.0–52.2, fluorescence wavelength intensity, respectively) [Figure 3]c. The intracellular levels of ROS were significantly reduced following treatment with 2, 4, and 10 μM BaP in combination with 25 mM taurine groups compared to the BaP groups from 38.0–18.2, 38.0–18.7, and 38.0–19.5, respectively [Figure 3]c. Indeed, co-treatment with taurine decreased ROS accumulation in BaP groups when compared to the BaP group [Figure 3]c.
Figure 3: Effect of BaP on the production of intracellular ROS and the protective effect of taurine in A549 cells. Cells were treated with 0 μM (untreated control) and 2–50 μM BaP, with or without 25 mM taurine for 24 h and the levels of ROS were assessed using DCFH-DA staining. (a) The intensity of ROS-associated Fluorescence increased upon BaP treatment cell. (b) Fluorescence intensity was decreased in cells treated with the combination of BaP and with taurine. (c) ROS levels were quantified and are presented as the mean ± SEM (N = 3). aP < 0.05 versus BaP groups; bP < 0.05 versus control group; cP < 0.05 versus treated with taurine alone. BaP: Benzo[α]pyrene, SEM: Standard error of the mean, ROS: Reactive oxygen species.

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Using flow cytometry, the fluorescence intensities corresponding to intracellular ROS levels were found to be 6.80 FU, 11.28 FU, 21.14 FU, and 18.51 FU following treatment with 2, 4, 10, and 50 μM, respectively, and 23.78 FU in the control group (data not shown). The intracellular levels of ROS in the four BaP groups were significantly higher than those in the control group. However, ROS levels were significantly decreased by co-treatment with taurine (data not shown). These data revealed that taurine significantly inhibited the intracellular levels of ROS, indicating that taurine protected A549 cells from BaP-induced damage.

Effect of taurine on benzo[α]pyrene-induced mitochondria-mediated apoptosis of A549 cells

MMP examination revealed that the intensity of MMP-associated fluorescence was gradually decreased in A549 cells treated with various concentrations of BaP (2, 4, 10, and 50 μM) when compared with the control group [Figure 4]a and [Figure 4]b, indicating that taurine restored fluorescence intensity [Figure 4]c and [Figure 4]d.
Figure 4: Effect of BaP on MMP and the protective effect of taurine in A549 cells. (a) Cells stained with rho with BaP 2 μM group. (c) The fluorescence intensity increased following co-treatment with 25 mM taurine damine 123 (red fluorescence) were treated with 0 μM (untreated control) and 2–50 μM BaP, showed a gradual decrease in fluorescence intensity. (b) Quantification of the results in A, which are expressed as mean Rh123 fluorescence intensity ± SEM (N = 3). aP < 0.05 versus untreated BaP group; bP < 0.05 versus treated. (d) Quantification of the results presented in C, which are expressed as mean Rh123 fluorescence intensity ± SEM (N = 3). aP < 0.05 versus BaP in the same groups; bP < 0.05 versus control group. BaP: Benzo[α]pyrene, SEM: Standard error of the mean, MMP: Mitochondrial membrane potential.

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Based on the results of flow cytometry and fluorescence microscopy, the early apoptosis percentages were 78%, 80%, 80%, and 22%, respectively, in cells treated with 2, 4, 10, and 50 μM BaP, respectively, when compared with the control group, especially 50 μM BaP caused late apoptosis [Figure 5]a. Furthermore, taurine significantly decreased early and late apoptosis [Figure 5]b. The TUNEL assay indicated that the percentage of apoptosis were 3.4%, 19.7%, 26.7%, 68.3%, and 79.3% in cells treated with 0 (control), 2 BaP, 4 BaP, 10 BaP, and 50 μM BaP, respectively [Figure 6]a. By contrast, BaP treatment caused DNA fragmentation, which was very prominent in cells treated with BaP for 24 h [Figure 6]b. These data show that BaP triggers mitochondrial damage, cell apoptosis, and DNA damage, but taurine can prevent these damages.
Figure 5: Effect of BaP on apoptosis and the protective effect of taurine in A549 cells. Cells were treated with 0 μM BaP (untreated control) and 2–50 μM BaP, in the presence or absence of 25 mM taurine for 24 h to detect cell apoptosis phase using Annexin V and PI fluorescence on a flow cytometer. (a) Quantification of the results presented in cells apoptotic phases following treatment with 0 μM (untreated control) and 2–50 μM BaP. (a) which are expressed as mean apoptosis ratio ± SEM (N = 3). (b) Quantification of the results presented in cells apoptotic phases following treatment with 0 μM (untreated control) and 2–50 μM BaP in the presence or absence of 25 mM taurine, which are expressed as mean apoptosis ratio ± SEM (N = 3). aP < 0.05 versus live cells in the same group; bP < 0.05 versus control group in early apoptosis; cP < 0.05 versus control group in late apoptosis. BaP: Benzo[α]pyrene, SEM: Standard error of the mean, PI: Propidium iodide.

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Figure 6: Effect of BaP on late apoptosis phase in A549 cells. Cells were treated with 0 μM (untreated control) and 2–50 μM BaP for 24 h to detect DNA fragmentation in apoptotic cells using TUNEL staining on a flow cytometer. (a) Data are expressed as mean TUNEL positive ratio ± SEM (N = 3). aP < 0.05 versus control group; bP < 0.05 versus treated with 2 μM BaP; cP < 0.05 versus treated with 4 μM BaP; dP < 0.05 versus treated with 10 μM BaP. (b) DNA laddering assay detected DNA fragmentation for DNA extract from X-ray irradiated A549 cells by 2–50 μM BaP treatment. Representative figure of the formation of DNA ladder after BaP treatment due to DNA damage. BaP: Benzo[α]pyrene, SEM: Standard error of the mean.

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Effect of taurine on pro-and anti-apoptotic mechanisms affected by benzo[α]pyrene treatment of A549 cells

Both caspase-3 and -7 are important apoptotic markers that cleave apoptosis substrates to induce the intrinsic apoptotic pathway and are activated in the late phase of apoptosis. The activation of caspase-3 and -7 was increased following treatment with BaP compared with the control group [Figure 7]. Moreover, the BaP + taurine group showed decreased caspase-3 and -7 activity when compared with the BaP group [Figure 7]. This result further showed that the apoptosis pathway induced by BaP is mediated by caspase-3 and -7, and taurine could inhibit caspase-3 and -7 activity.
Figure 7: Effect of BaP on caspase-3/7 activity and the protective effect of taurine in A549 cells. Cells were treated with 0 μM (untreated control) and 2–50 μM BaP, in the presence or absence of 25 mM taurine for 24 h and caspase-3/7 activity was assayed using FITC fluorescence on a flow cytometer. Data are expressed as mean caspase-3/7 expression ± SEM (N = 3). aP < 0.05 versus control group; bP < 0.05 versus treated with BaP in the same group. BaP: Benzo[α]pyrene, SEM: Standard error of the mean.

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Moreover, apoptosis via the caspase pathway induced by BaP was seen. qPCR and western blot analysis revealed that BaP increased Bax and Bak gene and protein expression, but taurine significantly decreased the levels of these genes and proteins when compared with those in the BaP group [Figure 8]a, [Figure 8]c, and [Figure 9]a, [Figure 9]c. In addition, cytochrome c protein expression was increased by different concentrations of BaP when compared with the control group, but taurine decreased expression of this protein when compared with the control group [Figure 9]a and [Figure 9]c. Moreover, Bcl-2, Bcl-x1, Bcl-2 gene and protein levels were decreased in the BaP groups when compared with those in the control group [Figure 8]b and [Figure 9]b. Taurine also increased Bcl-2 gene, Bcl-x1 protein, and Bcl-2 protein expression when compared with that in the control group [Figure 8]d and [Figure 9]d. NF-κB has both pro-apoptotic and anti-apoptotic functions. The expression of active phospho-65 of NF-κB and NF-κB protein levels were increased following treatment with different concentrations of BaP [Figure 9]b. However, taurine increased the levels of phospho-65 of NF-κB and NF-κB protein in cells treated with 10 and 50 μM BaP when compared with those in the control group [Figure 9]d. These data indicate that BaP triggers NF-κB activity to upregulate the caspase response and induce the apoptotic pathway.
Figure 8: Effect of BaP on the expression of pro-apoptotic genes, Bax and Bak, and anti-apoptotic gene, Bcl-2, and the protective effect of 25 mM taurine in A549 cells. Cells were treated with 0 μM (untreated control) and 2–50 μM BaP, in the presence or absence of 25 mM taurine for 24 h and gene expression was analyzed using real-time PCR. GAPDH was used as internal control. Expression of pro-apoptotic genes (a) and anti-apoptotic genes (b) following treatment with different concentrations of BaP. (c and d) Quantitative data on the expression of Bax, Bak and Bcl-2 genes following co-treatment with 25 mM taurine. The histograms indicate the mean ± SEM (N = 3). aP < 0.05 versus untreated BaP and taurine in Bax gene expression; bP < 0.05 versus untreated BaP and taurine in Bak gene expression; cP < 0.05 versus untreated BaP and taurine in Bcl-2 gene expression. BaP: Benzo[α]pyrene, SEM: Standard error of the mean, PCR: Polymerase chain reaction, Bcl-2: B-cell lymphoma-2, Bax: BCL-2-associated X protein.

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Figure 9: Effect of BaP on the expression of the pro-apoptotic proteins Bax and Bak and anti-apoptotic protein Bcl-2 and the protective effect of 25 mM taurine in A549 cells. Cells were treated with 0 μM (untreated control) and 2–50 μM BaP, in the presence or absence of 25 mM taurine for 24 h. Expression of pro-apoptotic proteins (a) and the anti-apoptotic protein (b) following treatment with different concentrations of BaP. The histograms indicate the mean ± SEM (N = 3). aP < 0.05 BaP-treated versus untreated regarding Bax and Bcl2 protein expression; bP < 0.05 BaP-treated versus untreated regarding Bak and Bcl-x1 protein expression; cP < 0.05 BaP-treated versus untreated regarding cytochrome c and NFκB protein expression. (c and d) protein expression following co-treatment with 25 mM taurine. aP < 0.05 untreated versus co-treated with taurine regarding Bax and Bcl2 protein expression; bP < 0.05 untreated versus co-treated with taurine regarding Cytochrome c and Bcl-x1 protein expression; cP < 0.05 untreated versus co-treated with taurine regarding NFκB protein expression. BaP: Benzo[α]pyrene, Bcl-2: B-cell lymphoma-2, Bax: BCL-2-associated X protein.

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  Discussion Top


BaP is recognized as a major toxic factor associated with the development of lung cancer worldwide. It has been reported that BaP induces the production of ROS, causing cell proliferation, apoptosis, inflammation, and DNA alterations. Taurine is rich in fish, especially in mussels and squids. Taurine is reported to act as a biofunctional substance that can prevent damage induced by ROS, heavy metals, and environmental pollution.

In this study, the effect of taurine on the toxicity of ROS was studied using the human pulmonary alveolar epithelial A549 cells. BaP induced cell cycle arrest and decreased cell viability in a dose-dependent manner. The levels of ROS and MMPs were elevated by 52.2% and 80%, respectively. Moreover, the expression of NF-κB, Bak, Bax, and cytochrome c at both the RNA and protein levels was increased, and the expression of Bcl-2 and Bcl-x1 was decreased by BaP treatment. BaP also activated caspase-3 and -7 expression.

However, taurine significantly reduced BaP-induced increase in ROS levels and restored the MMP. Taurine also decreased BaP-induced expression of NF-κB, Bak, Bax, and cytochrome c, activated caspase-3 and -7 activation, and increased the expression of Bcl-2 and Bcl-x1. It could be concluded that taurine ameliorates BaP toxicity in human pulmonary alveolar epithelial A549 cells. The other report revealed that BaP could increase carcinogenesis by decreasing the nonenzymic anti-oxidants and increasing the lipid peroxides to cause lung cancer in Swiss albino mice.[22] Meanwhile, taurine has an antioxidant and chemopreventive to attenuate the lung carcinogenesis by BaP-treatment in mice.[22] This result was similar to our finding. These evidence suggested that taurine can protect cell function to avoid cell apoptosis induced by high toxic chemicals. Nevertheless, the other report pointed out that high concentration of taurine probably suppressed the cell proliferation and enhanced the apoptosis through the upregulation of PUMA and Bax and downregulation of Bcl-2 in human lung cancer cell.[23] It means that high dose of taurine plays a inhibit function on a cancer cell in vitro study.

In previous studies,[10],[14] taurine was shown to block ROS generation and protect neural cell differentiation and cell apoptosis by reducing ER stress and mitochondrial dysfunction induced by aluminum and arsenite. In addition, taurine can prevent hepatic steatosis by modulating intracellular calcium homeostasis and energy metabolism.[24],[25] BaP has also been reported to trigger mitochondrial dysfunction, increase ROS levels, and induce cell apoptosis and DNA damage.[5],[10],[14] Therefore, BaP produces intracellular ROS levels, reduces MMP, and causes cell arrest.

NF-κB is an inducible transcription factor that plays an important role in regulating cell functions, such as cell inflammation, proliferation, apoptosis, morphogenesis, and differentiation.[26] In previous studies, BaP was reported to increase NF-κB expression in lung tissues and lung cancer cells.[27],[28] Moreover, the intrinsic apoptotic pathway involves ROS elevation to induce pro-apoptotic Bax and reduce anti-apoptotic Bcl-2 levels.[29] In this study, BaP was found to induce NF-κB protein expression via upregulation of Bax and Bak and downregulation of Bcl2 and Bcl-x1, which in turn triggered cytochrome c release by a caspase-3/7 mediated apoptotic pathway in A549 cells. In this study, taurine was found to reduce NF-κB protein expression to increase levels of Bcl2 and Bcl-x1 expression, decrease levels of Bax, Bak, and cytochrome c expression, and diminish caspase-3/7 activation. These findings verified the antioxidant effect of taurine in protecting against BaP-induced lung cell apoptosis.

Sikdar et al. reported that ROS caused MMP depolarization and triggered cytochrome c release.[30] Then, the permeability transition pore of the mitochondrial membrane opens to induce swelling of the mitochondria and rupture of the outer mitochondrial membrane. The activation of both Bak and Bax leads to mitochondrial outer membrane permeabilization, which is the defining event of the intrinsic apoptosis pathway and triggers caspase-3 and -7 activation to induce cell death.[8] Thus, it is suggested that BaP induces ROS accumulation, which results in increased cytochrome c expression, elevation of MMP depolarization, and mitochondrial-dependent apoptosis in A549 cells. This study showed that taurine reduced MMP depolarization and cytochrome c release and prevented cell apoptosis.

This study also showed that taurine reduced BaP-induced intracellular ROS levels, thereby decreasing mitochondrial stress and preventing the intrinsic pathway of apoptosis in lung cells. Hence, taurine reduced BaP-induced NF-κB protein expression and cytochrome c levels, upregulated Bcl-2 and Bcl-x1, and downregulated Bak and Bax expression at both RNA and protein levels through the caspase-3/7-mediated mitochondria-dependent apoptosis pathway via ROS in lung cells. Therefore, it can be concluded that BaP induces ROS production and enhances mitochondrial stress to cause intrinsic apoptosis, and that taurine plays a regulatory role in protecting against BaP-induced damage in A549 cells.


  Conclusions Top


The effect of taurine on the biotoxicology of BaP was tested using A549 cells. The results showed that BaP induced the intrinsic pathway of apoptosis in A549 cells by increasing caspase-3/7 activity and NF-κB protein expression. Taurine significantly inhibited these molecular changes and damages. Therefore, taurine plays a role in the attenuation of oxidative stress and mitochondrial dysfunction induced by BaP.

Financial support and sponsorship

We thank the Ministry of Science and Technology for supporting this research (grant number: 106-2314-B-019-002).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]



 

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