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Abstract

Benzo(a)pyrene (BaP) has been shown to be an inducer of apoptosis. However, mechanisms involved in BaP-induced mitochondrial dysfunction are not well-known. In this study, human fetal lung fibroblasts cells were treated with BaP (8, 16, 32, 64 and 128 μM) for 4 and 12 h. Cell viability, intracellular level of reactive oxygen species (ROS), total antioxidant capacity (T-AOC), mitochondrial membrane potential (ΔΨ_m) and cytochrome c release were determined. Changes in transcriptional levels of p53-dependent apoptotic genes (p53, APAF1, CASPASE3, CASPASE9, NOXA and PUMA) were measured. At time point of 4 h, BaP induced the intracellular ROS generation in 64 (p < .05) and 128 μM BaP groups (p < .01) but decreased the T-AOC activities in 32, 64 (p < .05 for both) and 128 μM BaP groups (p < .01). At time point of 12 h, ΔΨ_m significantly decreased in ≥32 μM BaP groups (p < .05 for all). Amount of mitochondrial cytochrome c significantly increased in 128 μM BaP group (p < .01). Transcriptional levels of CASPASE3, CASPASE9, APAF1 and PUMA were up-regulated in all BaP groups (p < .05 for all) and in ≥32 μM groups for NOXA (p < .05). But only in 16 μM BaP group a relatively little expression of p53 mRNA was observed (p < .05). The results indicate that in the earlier period BaP promoted the generation of excessive ROS and subsequently the mitochondrial depolarization, whereas transactivations of the p53-dependent apoptotic genes were significantly induced at the later period.

Keywords

benzo(a)pyrene oxidative stress mitochondrial dysfunction human fetal lung fibroblasts (MRC-5) cells

Introduction

Evidence is emerging that mitochondria play an essential role in cell life and cell death. In addition to their primary function of converting organic materials into cellular energy in the adenosine triphosphate (ATP) form, mitochondria as decision-makers¹ regulate cell death.^2,3 Mitochondria consist of two membranes, the outer membrane in close communication with the cytosol encloses the entire organelle; the folded inner membrane with the proteins necessary for the electron transport chain involves in energy transduction.⁴ Mitochondrial membrane permeabilization is a key event of the apoptotic process, it is regulated by pro- and antiapoptotic members of the Bcl-2 family.⁵ For instance, the pro-apoptotic proteins (Bax and Bak) of Bcl-2 family might promote mitochondrial outer membrane permeabilization, leading to the release of cytochrome c and other proteins (Smac/DIABLO).^6,7 Subsequently, the apoptosis signaling was initiated via the caspases.⁸ Whereas, the inner transmembrane potential is regulated by the mitochondrial permeability transition, which is as a result of calcium overload or oxidative stress.⁹ These changes affect the integrities of mitochondrial inner and outer membranes.¹⁰

The mitochondrial disruption is recognized by subtle, probably non-specific ultrastructural changes, such as the loss of the mitochondrial membrane potential (ΔΨ_m) and the increase of reactive oxygen species (ROS).¹¹ As the outer membrane of mitochondria becomes permeable, cytochrome c translocates to the cytosol¹² and binds to the apoptosis protease activation factor (APAF1) and pro-caspase-9, which results in activation of CASPASE9 and sequential activation of CASPASE3.^13,14 Therefore, the breakdown of the ΔΨ_m is often used as an indicator of the cellular viability because it is one of the earliest intracellular events that occur following the induction of apoptosis. In addition, mitochondria are also a major source of ROS, they can serve as its target during the apoptosis process.¹⁵ Many studies reveal that ROS can also change ΔΨ_m, which precedes cell death induced by various toxicants.^16,17

Benzo(a)pyrene (BaP) has often been used as an indicator for the carcinogenic polycyclic aromatic hydrocarbons present in the environment. It is considered carcinogenic to humans by International Agency for Research on Cancer (IARC).¹⁸ Its carcinogensis depends on enzymatic metabolism of benzo(a)pyrene to the ultimate mutagen, benzo(a)pyrene diol epoxide (BPDE).¹⁹ Experimental data suggested that mitochondria themselves may be cellular targets of BaP.^20–22 During the metabolic process of BaP, a large proportion of BaP-quinones (BPQs) are biologically produced by cytochrome P450 isozymes and peroxidases.^23,24 BPQs are highly chemically active compounds that are associated with the production of ROS.^25,26 Under normal conditions, endogenous ROS with relatively low concentrations is implicated in the regulation of cell growth.²⁷ Studies suggest that BaP-induced ROS production in mitochondria leads to the intracellular oxidant/antioxidant imbalance.^28,29 In addition to BaP-induced the disruption of mitochondrial membrane potential and the release of cytochrome c from mitochondria, BPDE, the major active metabolite of BaP, binds covalently to DNA, which triggers p53-dependent DNA damage response and the cell-cycle arrest or apoptosis.³⁰

p53 is a well-characterized transcription factor for many p53-responsive genes involved in DNA damage response, cell cycle control and apoptosis. Studies indicate that the transcriptional activation of Bax (a pro-apoptotic member of Bcl-2 family),³¹ the generation of ROS and transcriptional up-regulation of death receptors (Fas and DR5) are implicated in the p53-mediated response to DNA damage.³² However, the underlying mechanisms are still unclear.

To determine the effect of BaP on the mitochondrial function and the transcriptional regulations of p53 target genes involved in apoptosis in human fetal lung fibroblasts (MRC-5), the biochemical indices including total antioxidant capacity, cytosolic release of cytochrome c, intracellular level of ROS, membrane potential of mitochondria and mRNA levels of the p53 target genes were measured.

Materials and methods

Chemicals and reagents

Benzo(a)pyrene (BaP), dimethyl sulfoxide (DMSO) and 2,7-dichlorodihydrofluorescein diacetate (H₂DCF-DA) were purchased form Sigma-Aldrich (St. Louis, MO, USA). Minimum essential medium (MEM) was purchased from HyClone, Thermo Scientific (Beijing, China). Fetal bovine serum was obtained from Gibco Laboratories (Grand Island Biological Co., Grand Island, NY, USA).

Cell culture and treatment

MRC-5 cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China). They were cultured in MEM supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO₂. Cells were treated with BaP (8, 16, 32, 64 and 128 μM) or DMSO (solvent control, final concentration <.1%) in different time periods (4 and 12 h) when they had reached 75% confluence. To compensate for the lack of microsomal cytochrome P450 enzymes, S9 mix (final concentration: 3%) was added to the corresponding media to metabolize BaP.³³ DMSO with S9 mix (final concentration: 3%) serves as the control.

Measurement of cell viability

To assess the number of viable cells in the culture, the CellTiter-Glo luminescent cell viability kit (Promega Corporation, Madison, WI, USA) was performed based on the quantitation of cellular ATP. Briefly, MRC-5 cells were cultured in 96-well plates (5 × 10³ cells/well), then treated with BaP at the indicated concentrations or DMSO alone for 4 and 12 h. At the time points, 100 µL of CellTiter-Glo reagent was added to each well for 10 min. The mixture was transferred onto an opaque-walled 96-well plate. The luminescent signal was read on a microplate reader (Synergy 2, Bio-Tek Instrument, Inc., Winooski, VT, USA).

Measurement of intracellular ROS

To monitor the intracellular accumulation of ROS, the fluorescent dye, 2′,7′-dichlorofluorescein diacetate (H₂DCF-DA) was used. The assay was performed following the procedure described by Wan et al.,³⁴ with minor modifications. Briefly, MRC-5 cells were cultured in 96-well plates (1 × 10⁴ cells/well) and then treated with BaP at the indicated concentrations or DMSO alone for 4 and 12 h. At the time points, cells were washed once with phosphate-buffered saline solution (PBS, pH = 7.2) and then incubated at 37°C for 30 min in PBS (pH = 7.2) with 20 µM H₂DCF-DA (from a stock solution of 20 mM H₂DCF-DA in DMSO). Subsequently, cells were washed 3 times with PBS (pH = 7.2). The fluorescent intensity of 2′,7′-dichlorofluorescein (DCF) was detected in a fluorescence microplate reader (Synergy 2, Bio-Tek Instrument, Inc., Winooski, VT, USA) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Data are reported in relation to untreated cells values (control) and represented as the mean ± SD from three independent experiments.

Assessment of total antioxidant capacity

The total antioxidant capacity (T-AOC) is an early biochemical marker of oxidative stress. In this study, T-AOC was assayed with a commercial kit (Nanjing Institute of Jiancheng Biological Engineering, Nanjing, China). The spectrometric method was applied to evaluate T-AOC. In the reaction mixture, ferric ion was reduced by antioxidant reducing agents and stable color of the Fe²⁺-O-phenanthroline complex is produced. Briefly, MRC-5 cells were cultured in 6-well plates (1 × 10⁶ cells/well) and then treated with either BaP at the indicated concentrations or DMSO alone for 4 and 12 h. The supernatants were used to measure the T-AOC activities at the time points. The optical density was measured at 520 nm. Data are expressed as U/ml (1 T-AOC unit is defined as 0.01 increase of absorbance value (A520) in 1 min at 37°C).

Measurement of mitochondrial membrane potential

Disruption of ΔΨ_m is one of the earliest intracellular events that occur following induction of apoptosis. Change in the ΔΨ_m as an indicator of cellular viability reflects metabolic activity and integrity of the mitochondrial membrane. It reflects metabolic activity and integrity of the mitochondrial membrane. In this study, changes in ΔΨ_m were examined by monitoring the fluorescent dye rhodamine 123 (Rh123, Sigma-Aldrich Co. St. Louis, MO, USA). MRC-5 cells cultured in 6-well plates (4 × 10⁵ cells/well) were treated with BaP at the indicated concentrations or DMSO alone for 4 and 12 h, and then incubated with Rhodamine 123 (10 mg/ml) at 37°C for another 30 min. Cells were washed twice with PBS (pH = 7.2) and re-suspended in 500 μl PBS (pH = 7.2). The percentage of cells stained with Rh 123 was determined by Rh 123 fluorescence using a flow cytometer (BD Science, San Jose, CA, USA) at excitation wavelength of 488 nm and an emission wavelength of 530 nm. Data are expressed as percentage of the DMSO control and shown as the mean ± SD from three independent experiments.

Measurement of cytochrome c release

The release of mitochondrial cytochrome c into the cytoplasm either is an indicator of mitochondrial injury or is a good early marker for apoptosis. In this study, the cytosolic cytochrome c was measured using a commercial cytochrome c enzyme immunoassay kit (R&D Systems China Co., Ltd, Shanghai, China). According to the manufacturer's instructions, MRC-5 cells were plated in 24-well plates (1 × 10⁵ cells/well) for 24 h. The next day, cells were treated with BaP at the indicated concentrations or DMSO alone for 4 and 12 h. The supernatant was used to detect the activity of the cytosolic cytochrome c at 450 nm with a fluorescence microplate reader (Synergy 2, Bio-Tek Instrument, Inc., Winooski, VT, USA). The final cytochrome c concentration was obtained from a standard curve expressed as pg/ml of protein.

Isolation of RNA and quantitative real-time polymerase chain reaction

Total cellular RNA was isolated and evaluated as previously described.³⁵ Briefly, total RNA was extracted from the MRC-5 cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to manufacturer’s instructions. Reverse transcription (RT) was performed using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, MD, USA) according to the manufacturer’s instructions. The primers of the genes (Table 1) used in the experiments were retrieved from the National Center for Biotechnology Information GenBank (www.ncbi.nih.gov) and synthesized by Limited Company of TaKaRa Biotechnology (Dalian, China). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was carried out on the ABI prism 7900 Sequence Detection System (Applied Biosystem, Foster City, CA, USA) using SYBR^® Green qPCR SuperMix-UDG kit (Invitrogen, Beijing, China) according to the manufacturer’s protocol. Each reaction was run in a final volume of 10 μl, reaction mixtures contained 2 μl of the cDNA product (equal to 2 μg of the original total RNA), 2 nM each primer, 5 μl of 2× SYBR Green PCR Master Mix (Applied Biosystem, Foster City, CA, USA) and 2.6 μl RNA enzyme-free water. Each reaction was run in triplicate with appropriate negative controls. PCR conditions were as follows: initial denaturation step at 50°C for 2 min followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec. The melting curve analysis was performed for identifications of the genes. All samples were normalized to the β-actin values and data were analyzed by the 2^-ΔΔCT method.³⁶

Table 1.

Forward (F) and reverse (R) primer sequences used to perform real-time polymerase chain reaction (PCR) analysis of gene expression

Gene		Primer sequence	Product size (bp)
p53	F	CCAAGGCCTCATTCAGCTCTC	123
p53	R	TGCCCAACAACACCAGCTC	123
Caspase9	F	TTGCCCACACCCAGTGACA	129
Caspase9	R	AGAGTGAGCCCACTGCTCAAAGA	129
Caspase3	F	GACTCTGGAATATCCCTGGACAACA	143
Caspase3	R	CTGAGGTTTGCTGCATCGACA	143
Apfe1	F	AGAAATCCACACGGGCCATC	129
Apfe1	R	GCCACCTTTGAACGTGAGTCTG	129
Noxa	F	TGGAAGTCGAGTGTGCTACTCAA	91
Noxa	R	CAGAAGAGTTTGGATATCAGATTCAGA	91
Puma	F	GACCTCAACGCACAGTACGA	144
Puma	R	CTAATTGGGCTCCATCTCG	144
β-Actin	F	TGATGATATCGCCGCGCT	284
	R	GCCACACGCAGCTCATTGTA
	F	GCCACACGCAGCTCATTGTA

Statistical analysis

All data was processed with SPSS 12.0 for Windows (SPSS Inc., Chicago, IL, USA). Data are presented as the means ± SD. Data were subjected to one- (dose, time) and two-way (dose × time) ANOVA to determine the significance of the main factors and their interactions. When ANOVA revealed a significant effect of dose or a dose × time interaction, differences among treatment means were tested using posthoc Dunnett's test to compare means of the treatment groups with the control means, p < .05 was considered statistically significant.

Results

Effect of BaP on the cell viability

On the basis of the finding that effect of BaP on the cell viability, a two-way ANOVA revealed BaP had an effect of dose (p < .01), time (p < .01) and a time × dose interaction on the cell viability (p < .01). As shown in Figure 1, a concentration-dependent decrease in the cell viability only at 128 μM BaP group (with S9 mix) was observed at 4 h after treatment (p < .01). At 12 h after treatment, with the exception of 8 μM BaP group, the significant decreases in the cell viability were observed in the other BaP-treated groups (p < .01 for all); however, no significant changes in the cell viability were observed at 4 and 12 h after treatment of BaP at the indicated concentrations (without S9 mix, data not shown). The results indicated that dose- and time-dependent effects of BaP on MRC-5 cell viability were associated with the activation of BaP. Therefore, in the following experiments of this study, BaP-treated cells in the present of S9 mix serve as the experimental model.

Figure 1.

Effect of benzo(a)pyrene (BaP) on cell viability in human fetal lung fibroblasts (MRC-5) cells. Cells were treated with BaP (8, 16, 32, 64 and 128 μM) and dimethyl sulfoxide (DMSO; final concentration [v/v]: <0.1%) for 4 and 12 h. The cell viability was detected by CellTiter-Glo Kit. The data are represented as mean ± SD for triplicate cultures (**p < .01 versus the control).

Effect of BaP on the intracellular ROS generation

To estimate the intracellular ROS production in BaP-treated MRC-5 cells, an oxidation-sensitive fluorescent probe DCFH-DA was used as a sensitive cytosolic marker for oxidative stress. Two-way ANOVA of ROS concentrations revealed an effect of dose (p < .01), time (p < .01) and a time × dose interaction (p < .01). As shown in Figure 2, at 4 h after the initial BaP treatment, the mean intracellular DCF fluorescence intensity increased significantly at 64 and 128 μM groups (p < .05 for 64 μM BaP-treated cells; p < .01 for 128 μM BaP-treated cells). But at 12 h after treatment, the mean fluorescence intensities markedly decreased in the 16 μM BaP group compared with the control group (p < .05 for all).

Figure 2.

Effect of benzo(a)pyrene (BaP) on the reactive oxygen species (ROS) generation in human fetal lung fibroblast (MRC-5) cells. Cells were treated with BaP (8, 16, 32, 64 and 128 μM) and dimethyl sulfoxide (DMSO; final concentration [v/v]: <0.1%) for 4 and 12 h. The intracellular ROS level was detected by fluorescence intensities of the 2′,7′-dichlorofluorescin (DCF). The data are represented as mean ± SD for triplicate cultures (*p < .05 and **p < .01 versus the control).

Effect of BaP on the T-AOC activity

T-AOC is a sum of the activities of the various antioxidative substances that is commonly used as an indicator of antioxidant capacity. Two-way ANOVA of T-AOC revealed an effect of dose (p < .05), time (p > .05) and no time × dose interaction. As shown in Figure 3, at 4 h after BaP treatment, the T-AOC activities of MRC-5 cells decreased significantly at 32, 64 and 128 μM groups compared with the control group (p < .05 for 32 and 64 μM BaP-treated cells; p < .01 for 128 μM BaP-treated cells).

Figure 3.

Effect of benzo(a)pyrene (BaP) on the total antioxidant capacity (T-AOC) in human fetal lung fibroblasts (MRC-5). Cells were treated with BaP (8, 16, 32, 64 and 128 μM) and dimethyl sulfoxide (DMSO; final concentration [v/v]: <0.1%) for 4 and 12 h. The T-AOC was detected by the ferric reducing/antioxidant power assay. The data are represented as mean ± SD for triplicate cultures (*p < .05 and **p < .01 versus the control).

Effect of BaP on ΔΨ_m

ΔΨ_m reflect an intermediate, unstable state of mitochondria. Changes in ΔΨ_m may lead to mitochondrial dysfunction and the final execution of cell death. The measurement of ΔΨ_m is used to study signaling mechanisms involved in the initiation of the apoptotic cascade. Two-way ANOVA of ΔΨ_m revealed an effect of dose (p < .01), time (p < .01) and a time × dose interaction (p < .01). As shown in Figure 4, at 4 h after BaP treatment, there was no significant change in ΔΨ_m in all cells (p > .05 for all). However, at 12 h after treatment, ΔΨ_m significantly decreased (p < .05 for 32 μM BaP-treated cells, p < .01 for 64 and 128 μM BaP-treated cells).

Figure 4.

Effect of benzo(a)pyrene (BaP) on the mitochondrial transmembrane potential (ΔΨ_m) in human fetal lung fibroblast (MRC-5) cells. Cells were treated with BaP (8, 16, 32, 64 and 128 μM) and dimethyl sulfoxide (DMSO; final concentration [v/v]: <0.1%) for 4 and 12 h. The change in the ΔΨ_m was measured by monitoring the fluorescent cationic dye rhodamine 123. The data are represented as mean ± SD for triplicate cultures (*p < .05 and **p < .01 versus the control).

Effect of BaP on the cytochrome c release

To determine whether the release of cytochrome c from mitochondria occurs in response to BaP, the release of cytochrome c from the mitochondria in the MRC-5 cells was assessed by ELISA. Two-way ANOVA of cytochrome c revealed an effect of dose (p < .01), time (p < .01), not time × dose interaction. As shown in Figure 5, at 4 h after BaP treatment, no change in cytosol-induced release of cytochrome c from mitochondria was observed in all cells, compared with the control group (p > .05 for all). At 12 h after BaP treatment, a significant amount of mitochondrial cytochrome c was detected in 128 μM BaP-treated cells compared with the control group (p < .01).

Figure 5.

Effect of benzo(a)pyrene (BaP) on the cytochrome c released in human fetal lung fibroblast (MRC-5) cells. Cells were treated with BaP (8, 16, 32, 64 and 128 μM) and dimethyl sulfoxide (DMSO; final concentration [v/v]: <0.1%) for 4 and 12 h. The release of cytochrome c in the supernatants was determined by an enzyme-linked immunosorbent assay (ELISA). The data are represented as mean ± SD for triplicate cultures (**p < .01 versus the control).

Effect of BaP on the mRNA expression of apoptosis-related genes

To determine if transcriptional activations of the selected apoptosis-related genes including p53, CASPASE9, CASPASE3, APAF1, PUMA and NOXA were involved in response to BaP, we detected mRNA levels of these genes. Two-way ANOVA of mRNA expressions revealed an effect of dose (p < .05), time (p < .05) and a time × dose interaction (p < .05). After BaP treatment for 4 h, no changes in transcriptional levels of the genes were observed (data not shown). However, after BaP treatment for 12 h, as shown in Figure 6, mRNA expressions of CASPASE9, CASPASE3, APAF1 and PUMA were obviously up-regulated in all BaP-treated cells (p < .05 or p < .01); the up-regulation of NOXA was also observed in greater than or equal to 32 μM BaP-treated cells (p < .05 for all). But relatively little expression of p53 mRNA was observed only in 16 μM BaP-treated cells (p < .05).

Figure 6.

Analysis of benzo(a)pyrene (BaP)-induced mRNA expression of the genes in human fetal lung fibroblast (MRC-5) cells. Cells were treated with BaP (8, 16, 32, 64 and 128 μM) and dimethyl sulfoxide (DMSO, final concentration (v/v): <0.1%) for 12 h. Relative changes in mRNA expression of the genes were calculated by the 2^-ΔΔCT method. Data from three independent experiments are presented as the mean fold expression (2^-ΔΔCT). *p < .05 and **p < .01 represent genes that were significantly up- or down-regulated compared with the solvent control group.

Discussion

Genotoxic effects of BaP have been well known, but limited data are available on the mechanism of BaP-induced mitochondria damage at the molecular level. Studies reported that in MRC-5 cells, BaP induced apoptosis via JNK1/FasL and JNK1/p53 signals; and in MRC-5 SV2 cells (human fetal lung fibroblast line transfected with SV40), the metabolically activated BaP increased DNA ploidy and induced resistance to confluence-initiated cell death and morphological change.^37,38 However, another study reported that oxidative stress may not be an important risk factor for BaP (at 10 mM of up to 24 h)-induced injury.¹⁹ In our study, the increase in generation of ROS was at relatively early time point (4 h), which may induced by the metabolic activation products of BaP, when the rat liver homogenate fraction (S9 mixture) was used in the cells to offset the activities of the endogenous metabolic enzymes.

In recent years, several published reports have indicated that BaP induced either ROS generation during its metabolic process and DNA damage³⁹ or apoptosis in several cell models.^22,40,41 Oxidative stress occurs when there is an imbalance in the production and detoxification of reactive oxygen species within an organism. ROS as a signaling molecule can induce mitochondrial dysfunction and cell apoptosis.⁴² Excessive ROS lead to glutaredoxin oxidation, which then disassociates from signal-regulating kinase (ASK1) to initiate the activation of the c-Jun N-terminal kinase (JNK) signal transduction pathway.^43,44 JNK has an essential role in modulating the functions of pro- and antiapoptotic proteins located in the mitochondria. It causes release of mitochondrial cytochrome c and induces activation of the cytochrome c-mediated death pathway.^45,46 In our study, BaP induced the excessive intracellular ROS generation in a short period of time (4 h). These data are consistent with the previous reports.^47,48 Additionally, we observed the mitochondrial dysfunction resulting from the generation of excessive BaP-induced ROS. The results indicated that the mitochondrial dysfunction in BaP-treated MRC-5 cells may be mediated by ROS- and subsequently activate JNK-dependent apoptotic pathway. However, the antioxidant mechanisms remain to be further investigated.

Apoptosis is triggered by activated intracellular proteases named caspases during the onset of apoptosis by extrinsic and intrinsic pathways. The extrinsic pathway (receptor-mediated) involves receptor binding, followed by the initiator CASPASE8, which in turn activates CASPASE3, leading to the release of cytochrome c, the apoptosome formation and activation of CASPASE9. The intrinsic (mitochondrial) pathway involves members of the Bcl-2 family that regulate the cytochrome c release from mitochondria, followed by cytochrome c which can interact with APAF1, dATP and CASPASE9 to form apoptosome, which further activates procaspase3,⁴⁹ leading to apoptotic cell death. Previous studies demonstrated that BaP triggered the p53-mediated apoptosis cascade,^50,51 and mitochondria were involved during BaP-induced apoptotic process.⁵² A recent study reported that over-expressions of NOXA and PUMA indirectly promoted mitochondrial depolarization,⁵³ although the difference between PUMA-α and PUMA-β isoforms expression need further studies.⁵⁴ In our study, after treating MRC-5 cells with BaP for 12 h, obvious transcriptional up-regulations of apoptosis-related genes (CASPASE3, CASPASE9, APAF1, NOXA and PUMA) and the slight transcriptional up-regulation of p53, suggest that BaP-induced transcriptional expression of PUMA and NOXA may be modulated by p53-dependent or independent pathways, because the dependence of PUMA and NOXA induction on p53 was different in various cell types.⁵⁵ In addition, the transcriptional up-regulation of PUMA and NOXA in response to p53-independent apoptotic stimuli has been demonstrated.^56–58

In conclusion, the findings indicate that BaP in the MRC-5 cells was activated by S9 mix; there may be a link between the generations of excessive mitochondrial ROS and subsequently the mitochondrial membrane depolarization, mRNA expressions of the p53-responsive genes at a later time. Our study provides a model to further investigate the underlying mechanisms of BaP-induced mitochondrial dysfunction in MRC-5 cells.

Footnotes

This work was supported by funding from the National Natural Science Foundation of China (NNSFC, grant No. 30671735).

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Mitochondrial dysfunction and transactivation of p53-dependent apoptotic genes in BaP-treated human fetal lung fibroblasts

Abstract

Keywords

Introduction

Materials and methods

Chemicals and reagents

Cell culture and treatment

Measurement of cell viability

Measurement of intracellular ROS

Assessment of total antioxidant capacity

Measurement of mitochondrial membrane potential

Measurement of cytochrome c release

Isolation of RNA and quantitative real-time polymerase chain reaction

Statistical analysis

Results

Effect of BaP on the cell viability

Effect of BaP on the intracellular ROS generation

Effect of BaP on the T-AOC activity

Effect of BaP on ΔΨm

Effect of BaP on the cytochrome c release

Effect of BaP on the mRNA expression of apoptosis-related genes

Discussion

Footnotes

References

Effect of BaP on ΔΨ_m