Forchlorfenuron Exposure Induces Hepatocyte Apoptosis via MKK3/P38/ATF2 Pathway

Abstract

Forchlorfenuron is a widely used plant cytokinin in Traditional Chinese Medicine and agricultural cultivation to boost resistance, postpone senescence, and increase productivity. However, the improper use of forchlorfenuron results in excessive residues and contamination, raising health and safety concerns. Our research investigated the toxicity of forchlorfenuron on hepatocytes in vitro. Results showed that forchlorfenuron inhibited HepaRG cell viability in a concentration and time-dependent manner. Forchlorfenuron-induced cellular apoptosis and the increased intracellular reactive oxygen species (ROS) indicated the participation of oxidative stress. Molecular docking and network pharmacology data suggested that the hepatotoxicity of forchlorfenuron might involve the MAPK signaling pathway. After 24 h of forchlorfenuron exposure, the P38-MAP kinase, upstream kinases MKK3, and the transcription factor ATF2 were maximally activated. Apoptosis induced by forchlorfenuron was significantly reduced by pretreatment with the P38 inhibitor SB203580. These findings implicated that HepaRG hepatocyte injuries were generated by forchlorfenuron through the induction of cellular apoptosis via the MKK3/P38/ATF2 pathway. Forchlorfenuron application should be closely managed to prevent potential liver damage.

1. Introduction

Forchlorfenuron, 1-(2-chloro-4-pyridyl)-3-phenyl urea, is currently the most active cytokinin synthesized, which accelerates cell mitosis, promotes cell growth and differentiation, and prevents fruit falling [1]. Forchlorfenuron was usually used in the plantations of fruits and herbal medicines to increase fruit production and promote plant cell division [2,3]. However, increasing evidence has demonstrated that forchlorfenuron activates inflammation in the body, reduces steroidogenesis, and produces cytotoxicity [4]. Previous studies demonstrated that forchlorfenuron caused cytotoxicity, cytoskeleton destruction, and decreased expression of corresponding proteins in H9c2 cardiomyocytes in vitro. It also caused cardiac morphology deformation, cardiac contractile dysfunction, and erythrocyte reduction in zebrafish [5]. There is ample evidence to suggest the potential threat of forchlorfenuron to the cardiovascular system, but its toxic effects on other organs have not yet been demonstrated [6,7]. In this study, our research aims to explore the forchlorfenuron’s potential effective on the hepatocyte and illuminate the potential molecular mechanism.

Apoptosis is one of the most common forms of cell death in exogenous chemical-induced toxicity in the liver, which is modulated exquisitely by different intracellular protein kinase cascades [8,9]. Accumulating evidence indicates that an imbalance of mitochondrial membrane potential and oxidative stress from elevated ROS can induce cell apoptosis and may provide a means to target cancer cells [10]. Meanwhile, the activation of the Mitogen-activated protein kinase (MAPK) pathway is closely related to ROS and participates in cell apoptosis [11]. MAPK family proteins significantly regulate cell survival and death, including apoptosis. The extracellular signal-regulated kinases (ERKs) generally contribute to proliferation and growth, whereas P38 and c-jun N-terminal kinase (JNK) are involved in cell death [12]. A wide range of extracellular ligands and stresses trigger the activation of P38 MAPK. The P38 MAPK undergoes dual phosphorylation through the action of the upstream kinases MAPKK3 (MKK3) and MAPKK6 (MKK6) [13]. Upon activation, P38 phosphorylates various substrates in the cytoplasm and nucleus, including transcription factor 2 (ATF2) [14]. Bcl2 family members, including anti-apoptotic and pro-apoptotic proteins, have been reported to be involved in the mitochondrial pathway [15,16]. Bcl2/Bax ratios were crucial to determine whether the cell undergoes survival or apoptosis. Bcl2, one of the most potent inhibitors of apoptosis, contains a CRE element responsive to ATF2 in its promoter region.

In this study, we conducted to investigate the effects of forchlorfenuron on mitochondria and apoptosis in HepaRG cells and revealed the specific toxicological mechanism of forchlorfenuron-induced hepatic damage. Finally, we delve into the understanding of the reproductive toxicity of forchlorfenuron and provide future directions for drug therapy.

2. Results

2.1. Effects of Forchlorfenuron on HepaRG Cell Viabilities

MTT assays were used to investigate the cytotoxic effects of forchlorfenuron on HepaRG cells. Results showed that forchlorfenuron inhibited cell viability in a time-concentration manner (Figure 1). As shown in Supplementary S1, the IC50 values for HepaRG cells of forchlorfenuron at 24 h, 48 h, and 72 h were 149.5 μM, 109.6 μM, and 100.5 μM separately. Therefore, we used 200, 100, and 50 μM forchlorfenuron intervention for 24 h to further explore the toxicity mechanism of forchlorfenuron.

2.2. Forchlorfenuron Incubation Induced Cell Apoptosis in HepaRG Cells

Hoechst 33,342 staining and Annexin V-FITC/PI staining for flow cytometry analysis were conducted to explore the apoptosis induced by forchlorfenuron incubation. As shown in Figure 2A, normal HepaRG cells exhibited homogeneous fluorescence intensity of nuclei, while heterogeneous intensity and chromatin condensation of nuclei appeared in forchlorfenuron-treated cells. Quantitative analysis by flow cytometry showed that the apoptosis rate increased in a concentration-related manner (Figure 2B,C). Western blot experiment showed that the expression of pro-apoptotic protein Bax increased and the expression of anti-apoptotic protein Bcl2 decreased with the concentration of forchlorfenuron increased (Figure 2D,E).

2.3. Forchlorfenuron Regulated ROS Level in HepaRG Cell

DCFH-DA staining was used to explore the intracellular ROS level. As shown in Figure 3A,B, normal cells exhibited a low density of green fluorescence. After 24 h incubation of forchlorfenuron, there was a significant increase in intracellular ROS level, suggesting the induction of oxidative stress by forchlorfenuron.

2.4. Forchlorfenuron Induced the Depolarization of Mitochondrial Membrane Potential (Δψm) in HepaRG Cells

Mitochondrial membrane potential depolarization was considered the early sign of activation of the mitochondrial pathway. As shown in Figure 3C,D, in the control group, most cells exhibited red fluorescence, indicating the aggregated state of JC-1 inside mitochondria. Along with the increased concentration of forchlorfenuron, the number of cells with green fluorescence also increased, suggesting that forchlorfenuron caused the depolarization of mitochondrial transmembrane potential.

2.5. Network Pharmacology Analysis

The results of network pharmacology indicated that the liver injury caused by forchlorfenuron involved several pathways and targets. A total of 50,487 liver damage targets and 694 forchlorfenuron targets were found through database searches. Forchlorfenuron and liver injury were shown to be associated with 428 genes in total (Figure 4A). A protein–protein interaction (PPI) network was created by importing these 428 genes into the STRING database, and then the results were imported into the Cytoscape software (version 3.9.1) (Figure 4C). The Metascape website showed that the biological functions of 428 shared targets were revealed based on GO enrichment results in biological processes, cellular components, and molecular functions. The primary functions of the target genes were protein phosphorylation, MAPK cascade regulation, and protein kinase binding. The KEGG pathway results showed that the genes were significantly enriched in the MAPK signaling pathway, the PI3K-AKT signaling pathway, the Ras signaling pathway, and apoptosis. The PI3K-AKT signaling pathway and the MAPK signaling pathway, sorted by degree values, are circled in red as the first two pathways (Figure 4B).

2.6. Forchlorfenuron Bound Virtually to P38 and JNK

Molecular docking was used to establish the binding potential of forchlorfenuron to essential targets of the MAPK and PI3K-AKT signaling cascade. According to the molecular docking experiments, forchlorfenuron might bind virtually to P38 and JNK, the vital target proteins in the MAPK signaling cascade linked to apoptosis. Their binding energies were −7.06 kcal/mol and −6.03 kcal/mol. The binding energy of AKT protein to forchlorfenuron was larger than −5 kcal/mol, and its binding capacity was comparatively low (Figure 4D). In conclusion, forchlorfenuron might damage liver tissue through the MAPK signaling pathway based on the intermolecular interactions investigated by molecular docking and the predicted binding types and affinities of those interactions.

2.7. P38 MAPK Signaling Pathway Was Activated in Forchlorfenuron-Induced HepaRG Cells at 24 h

The MAPK pathway played a vital role in stress-induced apoptosis in liver cell injuries. To investigate the time course of MAPK pathway activation upon forchlorfenuron treatment, Western blot analysis was used to examine the protein expression of p-P38 and p-JNK at 1, 3, 6, 9, 24, and 48 h. Results (Figure 5) showed that both P38 and JNK pathways were activated upon forchlorfenuron incubation. The phosphorylation of P38 peaked at 24 h and decreased at 48 h, while the expression of p-JNK peaked at 48 h.

2.8. Forchlorfenuron Activated MKK3/P38/ATF2 Pathway in HepaRG Cells

To further confirm the role of the P38 MAPK pathway in forchlorfenuron hepatotoxic effects, we examined both the upstream regulator MKK3 and its downstream substrate ATF2. As shown in Figure 6, p-MKK3 and p-ATF2 expression increased in forchlorfenuron-treated HepaRG cells.

2.9. P38 Inhibitor SB203580 Can Inhibit Forchlorfenuron-Induced Cytotoxicity

As shown in Figure 7E, due to its toxic effects as a chemical substance and its intervention in P38 MAPK signaling in maintaining cellular homeostasis, SB203580 alone inhibited HepaRG cell viability, and forchlorfenuron reduced cell viability in a concentration-dependent manner [17,18]. Pretreatment with SB203580 reversed the cytotoxic effects of forchlorfenuron: cells in 100 μM and 50 μM groups pretreated with SB203580 exhibited significantly higher viabilities compared to cells treated with the same concentration of forchlorfenuron alone. Cells in 200 μM treated with and without SB203580 showed no difference in viability, which might be related to the severity of cell toxicities.

2.10. P38 Inhibitor SB203580 Can Inhibit Forchlorfenuron-Induced Apoptosis

Consistent with the results of cell viability, SB203580 alone induced HepaRG cell apoptosis, and the apoptosis rate in the 50 μM and 100 μM groups pretreated with SB203580 was significantly lower than in the group treated with forchlorfenuron alone. Moreover, when cells were pretreated with a P38 inhibitor, the pro-apoptotic protein Bax was inhibited, resulting in a decrease in the Bax/Bcl2 ratio by forchlorfenuron treatment. These results indicated that HepaRG cell apoptosis induced by forchlorfenuron may be directly related to the P38 MAPK pathway (Figure 7A,B).

3. Discussion

As a widely used plant growth regulator, the toxic effects of forchlorfenuron have attracted the attention of multiple researchers. The previous studies have demonstrated that forchlorfenuron induced inflammation, oxidative stress, and metabolic shifts in endometrial cancer cells. Compared with previous studies, our research focused on the toxic effects of forchlorfenuron on liver cells. Our study demonstrated that forchlorfenuron-induced HepaRG cells toxicity caused cellular apoptosis due to the increase in intracellular ROS and mitochondrial damage. The MAPK signaling pathway might be involved in forchlorfenuron-induced hepatotoxicity based on the results of network pharmacology and molecular docking. Synchronously, the cytotoxic effects of forchlorfenuron were further confirmed by cellular tests to link the MKK3/P38/ATF2 pathway. The application of the P38 inhibitor SB203580 reduced cellular damage, indicating the participation of the P38 MAPK pathway.

In this study, our results demonstrated that forchlorfenuron caused a significant increase in apoptosis, and the disruption of mitochondrial membrane potential, which indicated that forchlorfenuron activated the intrinsic pathway of apoptosis. Bcl-2 family proteins are important regulators of mitochondrial permeability [19]. Our results showed that forchlorfenuron decreased the expression of anti-apoptotic protein Bcl-2 and increased pro-apoptotic protein Bax expression, thus decreasing the Bcl2/Bax ratio. The interaction between oxidative stress and apoptosis has been profoundly investigated [20]. In our research, forchlorfenuron incubation caused increased ROS levels in HepaRG cells, indicating the involvement of oxidative stress in the hepatoxicity of forchlorfenuron.

Our network pharmacology analysis provided a predictive foundation for investigating the hepatotoxic mechanism of forchlorfenuron. The analysis identified a total of 428 overlapping targets between forchlorfenuron and chemical-induced liver injury. KEGG pathway enrichment analysis of these potential targets revealed significant associations with key pathways, including the MAPK signaling pathway, the PI3K-Akt signaling pathway, and apoptosis. The results of molecular docking experiments showed that forchlorfenuron has a higher binding energy with key proteins P38 and JNK in the MAPK pathway, thereby excluding the PI3K-Akt signaling pathway. Consistent with prior reports linking P38 and JNK activation to cellular apoptosis [21], our data confirm that both pathways were activated early after forchlorfenuron exposure. Notably, the phosphorylation of P38 peaked at 24 h and subsequently declined, whereas JNK activation peaked later, at 48 h. This earlier and pronounced activation of P38 temporally aligned with the key apoptotic events measured in our assays, suggesting it may play a more immediate role in initiating the cell death process under the present experimental conditions.

The MKK3 is a member of the dual-specificity protein kinase group that belongs to the MAPK signaling pathway, which phosphorylates specifically P38 MAPK upon activation. In recent research, MKK3 activation caused by oxidative stress stimulation has also been reported in other liver injuries [22]. In our research, the molecular docking outcomes and Western blot experiments demonstrated that forchlorfenuron incubation increased the activation of upstream p-MKK3 in a concentration-dependent way, which is consistent with the P38 MAPK phosphorylates [23] (Supplementary S2). Activated P38 MAPK phosphorylates a series of substrates such as transcription factor ATF2, which binds to specific target gene promoters and regulates target genes by histone modification. In our research, forchlorfenuron significantly increased p-ATF2 expression, thereby promoting hepatocyte apoptosis.

The use of P38 inhibitor SB203580 partially reduced the cytotoxicity in HepaRG cells, further confirming the role of the P38 pathway in the hepatotoxicity of forchlorfenuron. Cells in 100 μM and 50 μM forchlorfenuron groups are compared to cells pretreated with SB203580 and the same concentration of forchlorfenuron, respectively. The group that is pretreated with SB203580 significantly reversed the additional reduction in cell viability and the additional increase in apoptosis specifically induced by forchlorfenuron. However, when forchlorfenuron concentration reaches 200 µM, the P38 inhibitor SB203580 fails to rescue cells, with no significant improvement observed in apoptosis or viability rates. This is likely because the cellular damage induced by 200 µM forchlorfenuron is too severe and potentially involves mechanisms beyond or independent of the P38 pathway activation that SB203580 targets. Although SB203580 also caused cell damage when used alone, based on our experimental results, we can preliminarily conclude that forchlorfenuron partially activates the P38MAPK signaling pathway, leading to cell apoptosis.

However, two aspects of our study require some caution when interpreting the results. Firstly, these measurements were primarily conducted at a 24-h endpoint, which did not provide sufficient temporal resolution to determine the precise sequence of these molecular events. Although numerous studies have shown that the generation of ROS is often one of the early drivers and triggers of mitochondrial depolarization events and activation of p38-MAPK signaling pathway which induce apoptosis, especially under pathological conditions [24,25], the relationships among these three events are not simply in a sequential relationship, but form a positive feedback loop that jointly pushes cells towards the decision point of life and death [17,26]. Future research needs to clearly reveal the causal hierarchical relationships between these molecular events through more detailed time-series experiments and intervention experiments. Secondly, we had to admit that potential off-target effects of pharmacological inhibitors like SB203580 are a recognized limitation. The inherent effect of this P38 inhibitor might interfere with our conclusions [17,18]. Future studies employing genetic approaches would be valuable to provide further validation.

4. Materials and Methods

4.1. Materials

Forchlorfenuron was purchased from Sigma-Aldrich (St. Louis, MO, USA). MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, was a product of Sigma- Aldrich (St. Louis, MO, USA). Annexin V-FITC/PI Apoptosis kit was obtained from Sigma-Aldrich (St. Louis, MO, USA). P38 inhibitor SB203580 and primary antibodies (P38, p-P38, MKK3, p-MKK3, p-ATF2, ATF2, Bcl2, Bax) were bought from Cell Signaling Technology (Danvers, MA, USA).

4.2. Data Collection of Network Pharmacology

The Canonical SMILES of forchlorfenuron (CAS NO. 68157-60-8) was obtained from the PubChem website (https://pubchem.ncbi.nlm.nih.gov/). Then, the Swiss Target Prediction Database (http://www.swisstargetprediction.ch) and the PharmMapper database (https://www.lilab-ecust.cn/pharmmapper/submitfile.html) were used to collect potential targets for forchlorfenuron. “Chemical and Drug Induced Liver Injury” was designated as a keyword to search for hepatotoxicity targets on CTD websites (http://ctdbase.org/) [27]. The genes were standardized by using the Uniprot database (https://www.uniprot.org), and the intersecting targets were collected on the site venny 2.1 (https://www.biovenn.nl/index.php). The overlapped targets were integrated into the protein–protein interaction (PPI) network using the STRING database, and the minimum required interaction score was set to “≧0.9”. Then, the intersecting targets were imported into Cytoscape 3.9.1 software and Metascape (https://metascape.org/gp/index.html#/main/step1) to obtain the Gene Ontology (GO) functional annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively.

4.3. Molecular Docking

The file containing forchlorfenuron mol2 was obtained from the PubChem website. The PDB structures for the apoptotic pathway targets AKT, P38, MKK3, and JNK were obtained from the PDB database. Autodock 4.2.6 was used to create the molecular docking model, and Pymol was used to store the final result. The Autodock software allows one to store the Binding Energy data, export the docking file, perform molecular docking, hydrogenation, and charge operations on compounds and proteins, and then move on to visualization and analysis.

4.4. Cell Culture

HepaRG cells were obtained from Guan Dao Biotechnology (Shanghai, China) and cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin and 100 μg/mL streptomycin. The media and supplements were purchased from Gibco Life Technologies (Grand Island, NY, USA). The cell cultures were kept in a humidified atmosphere containing 5% CO₂ at 37 °C. Forchlorfenuron was dissolved in DMSO and incubated in an FBS-free complete medium with a final DMSO concentration of 1‰.

4.5. Cell Viability

Cell viability was determined using an MTT assay. Briefly, HepaRG cells were plated on 96-well plates at a density of 8 × 104 cells per well and then incubated at 37 °C for 24 h. The cells were treated with forchlorfenuron for 24 h, 48 h, and 72 h. MTT (5 mg/mL) was added to each well and incubated for four hours. The medium was removed, and the formazan crystals were dissolved with dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm on a microplate reader (TECAN Infinite M1000, Grödig, Austria).

4.6. Morphological Assessment and Quantification of Apoptotic HepaRG Cells

Hoechst 33,342 staining was used for the morphological analysis of the apoptosis of cells. Apoptotic cells exhibit nuclear chromatin condensation and fragmentation. After treatment, the cells were incubated with 5 mg/mL Hoechst33342 for 15 min, washed twice with phosphate-buffered saline (PBS), and visualized by fluorescence microscopy (Leica, Witzlar, Hesse, Germany).

4.7. Flow Cytometric Detection of Apoptosis

After the cells were treated with the indicated concentration of forchlorfenuron, the apoptosis rate was evaluated with the Annexin V-FITC/PI Apoptosis kit according to the manufacturer’s brochures. In brief, the cells were harvested, washed twice with cold PBS, incubated with the 5 μL FITC-Annexin V and 1 μL PI working solution (100 μg/mL) for 15 min in the dark at room temperature, and then cellular fluorescence was measured by flow cytometry analysis with a FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA).

4.8. Detection of Intracellular ROS Production

The production of intracellular ROS was analyzed using a Reactive Oxygen Species detection kit according to the manufacturer’s brochures (Invitrogen, Carlsbad, CA, USA). In conclusion, cells were washed with 1× wash buffer after treatment, and then the ROS detection solution was added. The cells were stained at 37 °C in the dark for 30 min and visualized by fluorescence microscopy (Leica, Witzlar, Hesse, Germany).

4.9. Determination of Mitochondrial Transmembrane Potential (Δψm)

5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) (Invitrogen, USA) was used to determine the changes in mitochondrial trans-membrane potential. Then, 10 μL of 200μM JC-1 (2 μM final concentration) was added to the cells, incubated for 30 min in the dark, and washed twice with PBS. The cells labeled with JC-1 were observed under fluorescence microscopy (Leica, Witzlar, Hesse, Germany).

4.10. Western Blot Analysis

Cultured HepaRG cells were harvested, washed with PBS, and lysed with cell lysis buffer containing 1% phenylmethylsulfonylfluoride. The lysate was centrifuged at 12,000 g for 15 min to remove the insoluble materials. Supernates were collected. Primary antibodies (P38, p-P38, JNK, p-JNK, MKK3, p-MKK3, p-ATF2, ATF2, Bcl2, Bax) from were used for indicated proteins.

4.11. Statistics

All data are expressed as the means ± SD of at least three independent experiments. Significant differences were determined by ANOVA, followed by Tukey’s multiple comparisons test between two groups. The level of significance was set at p < 0.05.

5. Conclusions

In conclusion, the plant growth regulator forchlorfenuron might cause in vitro hepatocyte toxicities. Mechanically, forchlorfenuron enhanced the intracellular ROS level in HepaRG cells, which induced apoptosis via activating the MKK3/P38/ATF2 pathway.