The effects of long-term co-exposure to nanocarbon black particles and benzo[a]pyrene on ROS levels and the AKT/P27 signaling pathway in BEAS-2B cells

Abstract

Background:

Long-term co-exposure to nanocarbon black particles (NCBP) and benzo[a]pyrene (B[a]P), as major components of PM2.5, may contribute to respiratory diseases by inducing oxidative damage. However, the molecular mechanisms underlying their synergistic effects remain unclear. This study aimed to investigate the impact of long-term co-exposure to NCBP and B[a]P on reactive oxygen species (ROS) levels and the protein kinase B (AKT)/cyclin-dependent kinase inhibitor 1B (P27) signaling pathway in human bronchial epithelial cells (BEAS-2B) cells.

Methods:

Cytotoxicity concentrations of NCBP and B[a]P for BEAS-2B cells were screened using the cell counting kit-8 assay and applied to subsequent long-term exposure experiments. Intracellular ROS levels were measured via fluorescent probe assay. Western blot was performed to analyze the expression of AKT/P27 pathway-related proteins (phosphatidylinositol 3-kinase, phosphorylated AKT [p-AKT], total AKT, and P27). The regulatory relationship between the pathway and P27 was validated using an AKT inhibitor, and statistical analysis was conducted to evaluate significance.

Results:

Significant reductions in cell viability (P < .05) were observed at concentrations of 16 μM B[a]P and 80 μg/mL NCBP. Long-term co-exposure to NCBP and B[a]P-induced ROS accumulation (P < .05). Western blot revealed upregulated p-AKT expression (P < .05) and significant suppression of P27 (P < .05) in the co-exposure group. Inhibition of p-AKT by an AKT inhibitor reversed the downregulation of P27 induced by co-exposure (P < .05), indicating that P27 is a downstream target of the AKT pathway. Compared to single exposures, co-exposure exhibited stronger synergistic effects on oxidative stress dysregulation.

Conclusion:

Long-term co-exposure to NCBP and B[a]P exacerbates oxidative damage in BEAS-2B cells by activating the AKT signaling pathway, suppressing P27 expression, and promoting ROS accumulation. This study elucidates potential mechanisms of multi-pollutant synergistic effects, providing new insights for health risk assessment of environmental co-exposure.

1. Introduction

The health risks associated with atmospheric particulate matter (PM2.5) have garnered increasing attention, with nanocarbon black particles (NCBP) and benzo[a]pyrene (B[a]P) identified as 2 principal components of PM2.5.^[1–3] NCBP are ultra-fine, lightweight black particulates formed from the incomplete combustion or thermal decomposition of hydrocarbons under oxygen-deficient conditions.^[4,5] Beyond their environmental presence, these particles also serve as crucial industrial nanomaterials, extensively utilized in the manufacture of products such as tires, rubber, and coatings.^[6] Due to their substantial surface area and strong adsorptive properties, these particles can absorb various other high-concentration pollutants. When suspended in the air, NCBP primarily enter the human body via inhalation, leading to respiratory and cardiovascular disorders. A notable response to their inhalation includes the increased expression of inflammatory markers in serum and lung tissues.^[7] Furthermore, research has demonstrated that exposure to NCBP can induce oxidative damage, mitochondrial dysfunction, and apoptosis in bronchial epithelial cells.^[8,9]

B[a]P, a ubiquitous polycyclic aromatic hydrocarbon, is prevalent in various environments, including air, soil, water, and fried foods, and is recognized as a significant pollutant with serious health implications.^[10–12] Extensive studies have shown that polycyclic aromatic hydrocarbons can cause severe pulmonary damage, including altered respiratory function, inflammation, fibrosis, and carcinogenesis.^[13,14] Moreover, B[a]P-induced lung damage has been linked to the dysregulation of several critical signaling pathways, such as the tumor protein p53, IL-6, and nuclear factor erythroid 2-related factor 2 pathways.^[15,16]

While existing research has established that both NCBP and B[a]P independently contribute to lung damage, studies on the long-term combined exposure of these substances remain scarce, and the specific mechanisms underlying their synergistic effects on lung injury are still unclear.^[17] Addressing this gap, the present study aims to explore the impact of long-term co-exposure to NCBP and B[a]P on oxidative damage and related signaling pathways in normal human bronchial epithelial cells (BEAS-2B). By investigating the combined effects of these pollutants on oxidative stress responses and signaling pathway regulation, this study seeks to provide new scientific insights into the mechanisms driving lung injury under conditions of environmental co-exposure.

2. Materials and methods

2.1. Main instruments and reagents

The primary equipment used in this study includes a CO₂ incubator (Thermo, USA), a full-wavelength microplate reader (Molecular Devices, USA), a fluorescence microscope (ZEISS, Germany), an ultracentrifuge (Herolab, Germany), a vortex mixer (IKA, Germany), a chemiluminescence imaging system (Bio-Rad, USA), and an electrophoresis system with transfer tanks (Bio-Rad, USA). The reagents employed include nanocarbon black particles (99.5%, 30 nm, Macklin Biochemical Technology Co., Ltd., Shanghai), AKT-IN-12 (MCE, Shanghai), 3,4-benzopyrene (96%, Macklin Biochemical Technology Co., Ltd., Shanghai), DMEM culture medium (Hyclone, USA), trypsin (Gibco, USA), fetal bovine serum (FBS, Gibco, USA), cell counting kit-8 (CCK-8) assay kit (Beyotime Biotechnology Co., Ltd., Shanghai), RIPA cell lysis buffer (Beyotime Biotechnology Co., Ltd., Shanghai), BCA protein quantification kit (Beyotime Biotechnology Co., Ltd., Shanghai), reactive oxygen species (ROS) assay kit (Beyotime Biotechnology Co., Ltd., Shanghai), rabbit anti-phosphatidylinositol 3-kinase (PI3K) antibody (CST, USA), rabbit anti-p-AKT antibody (CST, USA), rabbit anti-AKT antibody (CST, USA), rabbit anti-P27 antibody (CST, USA), and rabbit anti-β-actin antibody (CST, USA). The ECL chemiluminescence detection kit was also procured from Beyotime Biotechnology Co., Ltd., Shanghai.

2.2. Cell culture and preparation of test substances

BEAS-2B cells, stored in liquid nitrogen and provided by the Department of Occupational Health at Harbin Medical University, were revived and cultured in DMEM medium containing 10% FBS and 1% antibiotics. The cells were incubated at 37 °C in a 5% CO₂ incubator and subcultured when they reached 80% to 85% confluence using trypsin digestion.^[18] The cells were passaged 2 to 3 times after resuscitation and then subjected to subsequent exposure experiments.

NCBP were accurately weighed using an electronic balance and sterilized under UV light for 24 hours. After sterilization, the particles were dispersed in sterile PBS and ultrasonicated to ensure homogeneity (final concentration: 50 mg/mL). Similarly, B[a]P was sterilized under UV light and dissolved in a specific volume of DMSO to prepare a homogeneous solution (final concentration: 100 mM).

2.3. CCK-8 assay for cell viability

After trypsinization, BEAS-2B cells were resuspended in fresh medium, and the cell count was determined using a cell counter. Cells were seeded into 96-well plates at a density of 5 × 10³ cells/well and incubated at 37 ℃ in a 5% CO₂ incubator for 12 hours to allow adherence. The cells were then exposed to varying concentrations of NCBP (0 μg/mL, 10 μg/mL, 20 μg/mL, 40 μg/mL, 80 μg/mL, and 160 μg/mL) and B[a]P (0 μM, 2 μM, 4 μM, 8 μM, 16 μM, and 32 μM) for 24 hours. After exposure, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for an additional 2 hours. And blank control wells containing only DMEM and FBS were established. Absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated accordingly.

2.4. Measurement of intracellular ROS levels

BEAS-2B cells were seeded into culture dishes and divided into 4 groups: control, nanocarbon black exposure, benzo[a]pyrene exposure, and combined exposure. After adherence, the cells were subjected to repeated exposures over 14 passages. Following the ROS assay kit instructions, the cells were incubated with DCFH-DA probe for 30 minutes, and unincorporated probes were washed off with serum-free medium. Fluorescence was observed and photographed using a fluorescence microscope. The cells were then trypsinized, counted, transferred to 96-well plates at a density of 1 × 10⁴ cells/well. Fluorescence intensity was measured using a fluorescent microplate reader (excitation wavelength: 488 nm, emission wavelength: 525 nm).

The cells were incubated with 10 μM DCFH-DA in serum-free medium for 30 minutes, followed by 3 PBS washes. Subsequently, they were stained with 10 μg/mL Hoechst 33,342 and incubated at 37 °C for 10 minutes.

2.5. Western blot analysis of protein expression

BEAS-2B cells were subjected to 14 passages of exposure and then washed 3 times with precooled PBS. Cells were harvested by scraping, centrifuged to collect the pellet, and lysed in RIPA buffer on ice for 50 minutes. Protein concentrations were determined using the BCA method, and samples were denatured at 100 °C for 10 minutes. Proteins (20 μg of total protein per lane) were separated by SDS-PAGE and transferred onto PVDF membranes, which were blocked with BSA at room temperature for 3 hours. The membranes were then incubated with primary antibodies (PI3K, 1:800; p-AKT, 1:1000; AKT, 1:800; P27, 1:800; β-actin, 1:2000) overnight at 4 °C. After washing with TBST, the membranes were incubated with secondary antibodies (1:3000) at room temperature for 2.5 hours. Following further washes with TBST, the membranes were developed using ECL chemiluminescence, and band intensity was quantified using Image Lab software. The relative expression levels of PI3K, p-AKT, AKT, and P27 were normalized to β-actin. The methodology employed in the AKT inhibitor group is in accordance with that of the preceding group.

2.6. Data analysis

Statistical analysis of experimental data was performed using SPSS software. One-way ANOVA followed by Dunnett t test was used to compare differences between groups. Results were considered statistically significant at P < .05 and are presented as mean ± standard deviation (x̄±s).

3. Results and discussion

This study investigated the effects of long-term co-exposure to NCBP and B[a]P on ROS levels and the AKT/P27 signaling pathway in BEAS-2B cells, providing preliminary insights into their potential cytotoxic mechanisms. The results demonstrated that long-term co-exposure significantly increased intracellular ROS generation, activated the AKT signaling pathway, and suppressed P27 protein expression. These changes may collectively contribute to oxidative damage and abnormal cell cycle progression.

3.1. Cell viability

Low concentrations of B[a]P and nanoscale carbon black particles do not affect the viability of BEAS-2B cells. Cell viability begins to significantly decrease when the concentration of B[a]P reaches 16 μM. Similarly, cell viability starts to significantly decline when the concentration of nanoscale carbon black particles reaches 80 μg/mL, as shown in Figure 1. Therefore, for the subsequent long-term exposure experiments, we selected concentrations of 8 μM B[a]P and 40 μg/mL nanoscale carbon black particles.

Figure 1.

The CCK-8 assay was utilized to assess the impact of B[a]P(B[a]P)/nanoscale carbon black particle (NCBP) exposure on the viability of BEAS-2B cells. (A) Cell viability of BEAS-2B under various concentrations of B[a]P. (B) Cell viability of BEAS-2B under various concentrations of NCBP. Data were presented as mean ± SD (n = 6). *P < .05, **P < .01 compared to the control group. BEAS-2B = human bronchial epithelial cells, B[a]P = benzo[a]pyrene, CCK-8 = cell counting kit-8.

3.2. ROS generation and oxidative stress

Oxidative stress has been identified as a key mechanism driving nanoparticle-induced toxicity in both in vivo and in vitro systems. To elucidate how co-exposure impairs BEAS-2B cell proliferation, we further analyzed ROS-mediated signaling pathways, Our findings revealed that the long-term co-exposure to NCBP and B[a]P markedly elevated ROS levels in BEAS-2B cells.as shown in Figure 2. Consistent with prior evidence, nanoparticles leverage their high surface area and adsorption capacity to intensively bind to cell membranes, undergo endocytosis, and subsequently localize in mitochondria. This process drives ROS overproduction, oxidative stress activation, and ultimately mitochondrial dysfunction.^[19] B[a]P, as a polycyclic aromatic hydrocarbon, can also be metabolically activated to produce ROS, resulting in oxidative damage within cells.^[20,21] The excessive accumulation of ROS triggers a cascade of cellular responses, including mitochondrial dysfunction, DNA damage, and protein modifications, ultimately leading to apoptosis or necrosis.^[22]

Figure 2.

(A) Fluorescence microscopy images of cells exposed to 8 μM B[a]P and 80 μg/mL NCBP, with green fluorescence indicating ROS and blue indicating cell nuclei stained with Hoechst 33,342. (B) Fluorescence intensity was measured using a fluorescent microplate reader (excitation wavelength: 488 nm, emission wavelength: 525 nm). Relative fluorescence intensity of each group. Results are presented as mean ± standard deviation (n = 3). *P < .05; **P < .01 compared to the control group. B[a]P = benzo[a]pyrene, NCBP = nanocarbon black particles, ROS = reactive oxygen species.

3.3. Activation of the AKT signaling pathway and suppression of P27 protein expression

The expression levels of AKT signaling pathway-related proteins and P27 protein in cells exposed to 14 generations of B[a]P and NCBP are shown in Figure 3A–D. The expression of phospho-AKT (p-AKT) protein significantly increased in the group co-exposed to B[a]P and NCBP, while the expression level of P27 protein significantly decreased.

Figure 3.

Western blot experimental results. (A–B) Images and relative expression levels of PI3K protein, AKT protein, and p-AKT protein bands in different exposure groups.**P < .01, comparison between the co-exposure group of B[a]P and NCBP and the control group. (C–D) Images and relative expression levels of P27 protein bands in different exposure groups.**P < .01, comparison between the co-exposure group of B[a]P and NCBP and the control group. (E–F) The effect of AKT inhibitors on the expression levels of p-AKT and P27 proteins in the co-exposure group of B[a]P and NCBP. Results are presented as mean ± standard deviation (n = 3). **P < .01. AKT = protein kinase B, B[a]P = benzo[a]pyrene, NCBP = nanocarbon black particles, P27 = cyclin-dependent kinase inhibitor 1B, p-AKT = phosphorylated AKT, PI3K = phosphatidylinositol 3-kinase.

To further validate the relationship between the AKT signaling pathway and P27 protein during the co-exposure process of B[a]P and nanoscale carbon black particles, we conducted experiments using an AKT inhibitor (AKT-IN-12). The results are depicted in Figure 3E–F. The AKT inhibitor group can reduce AKT phosphorylation, leading to a decrease in p-AKT expression levels. We also observed that as the expression of p-AKT declined, there was a corresponding increase in P27 expression levels. This indirectly suggests that during the co-exposure of BEAS-2B cells to B[a]P and NCBP, P27 functions as a downstream protein of p-AKT.

The AKT signaling pathway plays a crucial role in regulating cell survival and proliferation.^[23] In this study, long-term co-exposure significantly activated the AKT pathway, which may represent a protective response by cells to oxidative stress. Once activated, AKT can promote the expression of downstream anti-apoptotic molecules, thereby enhancing cell survival. However, prolonged activation of AKT may also lead to uncontrolled cell proliferation and an increased risk of tumorigenesis. Our results align with other studies on nanoparticles and polycyclic aromatic hydrocarbons, supporting the critical role of ROS in the activation of the AKT signaling pathway.^[24]

P27, as a cell cycle inhibitor, plays a pivotal role in controlling cell cycle progression. A reduction in P27 expression facilitates cell cycle progression, thereby enhancing cell proliferation potential.^[25] In this study, we observed significant suppression of P27 expression under long-term co-exposure, which may be a direct consequence of AKT pathway activation. AKT phosphorylates P27, causing its translocation from the nucleus to the cytoplasm, where it is degraded, thus lifting the inhibition on Cyclin/CDK complexes. While this regulatory mechanism may provide short-term adaptive benefits in response to external stress, prolonged suppression of P27 could lead to abnormal cell cycle progression and an increased risk of tumor development.

Compared to single exposures, long-term co-exposure to NCBP and B[a]P exhibited more pronounced cytotoxic effects. This suggests that the co-exposure may exert synergistic effects, amplifying cellular damage. Possible mechanisms include the enhanced adsorption of B[a]P by NCBP, increasing its intracellular concentration, or the co-activation of more intense oxidative stress responses. This synergistic effect highlights the complexity of multiple pollutants coexisting in the environment, underscoring the need for greater attention to the health risks associated with multi-pollutant exposures.

4. Limitations and future directions

While this study provides valuable preliminary data, there are several limitations. First, the experiments were conducted exclusively in vitro, and these findings have not yet been validated in in vivo models. Second, although we explored the interrelationships between ROS, AKT, and P27, we have not fully elucidated the dynamic changes in these pathways over time. Additionally, other potential signaling pathways, such as nuclear factor erythroid 2-related factor 2 and tumor protein p53, may also be involved in the cellular responses to co-exposure, and future research should consider a systematic analysis of these pathways.

5. Conclusion

In summary, this study reveals the potential mechanisms by which long-term co-exposure to NCBP and B[a]P induces oxidative damage and abnormal cell cycle progression in BEAS-2B cells, through the enhancement of ROS generation, activation of the AKT signaling pathway, and suppression of P27 expression. These findings provide new insights into the health impacts of co-exposure to multiple environmental pollutants and lay the groundwork for future mechanistic studies and the development of protective strategies.

Author contributions

Conceptualization: Wei Zhang.

Data curation: Jiping Li, Lingxin Kong, Feng Hu, Hong Liang.

Methodology: Jiyuan Li.

Software: Shuli Ma.

Validation: Mingxia Wang.

Visualization: Wei Zhang, Linlin Du, Xingsan Li.

Writing – original draft: Jiping Li.

Writing – review & editing: Jiping Li.