Effects of Fludioxonil on the Cell Growth and Apoptosis in T and B Lymphocytes

Fludioxonil is fungicide used in agriculture, which is present in fruits and vegetables. In this study, the effects of fludioxonil on human immune cell viability, apoptosis, cell cycle arrest, and mitochondrial membrane potential were examined in human immune cells, such as Jurkat T cells and Ramos B cells. To examine the cell viability, Jurkat T cells and Ramos B cells were treated with fludioxonil (10−9–10−5 M) for 24 h and 48 h. Water soluble tetrazolium salt assay showed that fludioxonil decreased Jurkat T cell and Ramos B cell viability. Jurkat T cell viability decreased at 24 and 48 h, but Ramos B cell viability decreased only at 48 h. JC-1 dye revealed decreased mitochondrial membrane potential in fludioxonil-treated Jurkat T cells and Ramos B cells. To evaluate apoptosis, annexin-V conjugated FITC, AF488, and propidium iodide (PI) were used and to evaluate cell cycle arrest PI was used. Apoptosis and cell cycle arrest were induced by fludioxonil (10−7–10−5 M) in the Jurkat T cells at 24 and 48 h and Ramos B cells at 48 h. Moreover, the protein levels of pro-apoptotic proteins, such as p53, BAX, and cleaved caspase 3, were increased and anti-apoptotic protein Bcl-2 was decreased by fludioxonil. Expression of the Fas receptor related to the extrinsic apoptosis pathway was increased by fludioxonil. Additionally, cyclin D1 and cyclin E1 were decreased by fludioxonil. In the present study, fludioxonil induced immunotoxicity in human T cells and B cells through apoptosis and cell cycle arrest. Therefore, the present study suggests that fludioxonil induces the cellular toxicity in immune cells.


Introduction
Pesticides are chemical substances that kill living fungi, weeds, insects, and rodents. Though pesticides benefit human life via increasing agricultural productivity through protecting crops from fungi, weeds, and insects, etc., pesticides are toxic to humans and theenvironment. Many pesticides have been used in agriculture, such as organochlorines (OCs), organophosphorus (OPs), carbamate (CBs), pyrethroids, chlorophenoxys, triazines amid, and phthalimides. These pesticides remain in the air, water, and soil [1,2]. According to human health issues on pesticides released by the Environmental Protection Agency (EPA) of the US, they present that residual pesticide exposure occurs through the skin, eyes, inhalation, and ingestion. Residual pesticides, exposed to humans, are a hazard to human health, and can lead to disorders in nerves, dysfunction of the immune system, and endocrine disruption [3,4]. Additionally, pesticides are known to be harmful in humans, but are frequently used in agriculture.
Fludioxonil is a phenylpyroll fungicide and is an antifungal antibiotic, produced in 1993 by Sygenta [5]. Fludioxonil is used for cereals, fruits, and vegetables. It has a broad spectrum of activity [6]. Additionally, fludioxnil inhibits transport-associated phosphorylation of glucose that decreases mycelial growth [7]. Fludioxonil showed endocrine disruptor activity in androgen receptors

Treatment of Fludioxonil
The human Jurkat T cells and Ramos B cells were seeded at a density of 6 × 10 5 cells/mL in the 24-well plates (SPL Life Science, Pocheon, Republic of Korea) and 10 7 cells/mL in 90 mm cell culture dish (SPL Life Science). The seeded cells in 24-well plate were used to analyze apoptosis, cell cycle arrest, and mitochondrial membrane potential, and seeded cells in 90 mm dish were used for the extraction of proteins for Western blot analysis. Then, T cells and B cells were treated by fludioxonil at 10 −7 -10 −5 M for 24 or 48 h and incubated at 37 • C in 5% CO 2 .

Water Soluble Tetrazolium Salt (WST) Assay
The human Jurkat T cells, Ramos B cells, and WI-26 cells were seeded at a density of 5 × 10 4 cells/well in 200 µL in 96 well plates (SPL Life Science), and incubated in a humidified 5% CO 2 atmosphere at 37 • C for 24 h. Incubated cells were treated with various concentrations of fludioxonil (final concentration 10 −9 -10 −5 M in the medium) for 24 or 48 h. After 24 and 48 h, the cell viability was evaluated using EZ-Cytox (Daeil Lab, Seoul, Republic of Korea). EZ-Cytox 20 µL was added to each well and incubated at 37 • C for 1 h. The absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA).

FACS Analysis of Cell Cycle Arrest by PI Staining
In the 24-well plate, Jurkat T cells and Ramos B cells treated with fludioxonil (10 −7 M-10 −5 M) for 24 and 48 h were collected to 15 mL tubes, and collected cells were centrifuged at 400× g for 10 min and removed the supernatant. The cells were washed by phosphate-buffered saline (PBS) and resuspended by PBS, then the Jurkat T cells and Ramos B cells were fixed by 70% ethanol and the cells were incubated in 4 • C for 1 h. After cells were fixed, these cells were washed by PBS and stained with propidium iodide (PI) premix, which combined PI (Sigma-Aldrich Corp.) and RNase A (iNtRON Biotechnology, Seongnam, Republic of Korea). Then, cells, treated with PI, incubated at 4 • C for overnight. These cells, stained with PI and fixed with ethanol, were analyzed by flow cytometry (SH-800; Sony Biotechnology Inc., Bothell, WA, USA) to evaluate cell cycle arrest. A total of 30,000 cells were analyzed by flow cytometry.

FACS Analysis of Apoptosis by FITC, AF488-Annexin V/PI Staining
In the 24-well plate, Jurkat T cells and Ramos B cells were treated with fludioxonil (10 −7 M to 10 −5 M) for 24 and 48 h. The treated Jurkat T cells and Ramos B cells were collected to 15 mL tubes, and stained with FITC, Alexa Fluor 488-annexin V/PI (Invitrogen, Carlsbad, CA, USA) for 30 min. After staining, Jurkat T cells and Ramos B cells were washed by PBS. Then the stained Jurkat T cells and Ramos B cells were counted and 30,000 cells were analyzed by flow cytometry. Early apoptotic cells were defined as positive annexin V/negative PI and late apoptotic cells were defined as positive annexin V/positive PI.

Analysis of Mitochondrial Membrane Potential
In the 24-well plate, the immune cells were treated with fludioxonil (10 -7 M-10 -5 M) for 24 and 48 h and collected each 15 mL tubes, centrifuged at 400× g for 10 min, and the supernatant was removed. Remaining cells in the 15 mL tubes were stained by JC-1 dye (Invitrogen, Carlsbad, CA, USA) 10 µg/mL at 37 • C in 5% CO 2 incubator for 15-30 min. The stained cells were washed by PBS twice and resuspended in media. The stained cells were seeded in 0.2% gelatin-coated 24-well plates, and stained cells were incubated at 37 • C in 5% CO 2 overnight. The stained cells were observed with an Olympus CKX 41 microscope (Olympus Corp., Tokyo, Japan) under green fluorescence and red fluorescence. The red fluorescence indicates healthy mitochondria and the green fluorescence indicate depolarized membrane potentials of mitochondria.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from human Jurkat T cells and Ramos B cells using TRIzol Reagent (Invitrogen Life Technologies, Waltham, MA, USA). After isolating RNA, cDNA was synthesized using moloney murine leukemia virus reverse transcriptase (M-MLV RT) (iNtRON Biotechnology) from the isolated mRNA. Fas gene was amplified from cDNAs of Jurkat T cells and Ramos B cells to confirm gene expression levels. In this analysis, to compare Fas gene expression, the human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as an internal control as shown Table 2. To analyze the PCR product on 1.5% agarose gel containing NEOgreen (NEO Science, Suwon, Republic of Korea), electrophoresis was used. The bands representing the target genes were detected by LuminoGraph II (ATTO). Table 2. Oligonucleotide sequences of the semi-quantitative RT-PCR products.

Fludioxonil Exposure Supressed Cell Viability
The effects of fludioxonil on the viability of Jurkat T cells and Ramos B cells were measured by WST assay. A decrease in cell viability was observed after exposure of Jurkat T cells to fludioxonil for 24 and 48 h. In Jurkat T cells treated with fludioxonil for 24 h, cell viability was decreased by only a high concentration (10 −5 M). Additionally, in Jurkat T cells treated with fludioxonil for 48 h, cell viability was decreased by dose dependent in 10 −8 -10 −5 M ( Figure 1A). However, in the Ramos B cells treated with fludioxonil for 24 h, cell viability did not change, and in Ramos B cells treated with fludioxonil for 48 h, cell viability was decreased by of fludioxonil at concentrations of 10 −7 -10 −5 M in a dose-dependent manner ( Figure 1B). In WI-26 cells, which are human lung fibroblasts, treated with fludioxonil (10 −9 -10 −5 M) for 24 and 48 h, cell viability did not change ( Figure 1C).

Fludioxonil Exposure Induced Cell Cycle Arrest
Flow cytometry was used to confirm the changes in cell cycle induced in Jurkat T cells and Ramos B cells that were control or exposed to fludioxonil (10 −7 -10 −5 M) for 24 or 48 h. To confirm the effects on the cell cycle by fludioxonil, Jurkat T cells and Ramos B cells were stained by PI. Separation of cells in G0/G1, S phase, and G2/M was based on the linear fluorescence intensity after PI. In Jurkat T cells treated with fludioxonil (10 −7 -10 −5 M) for 24 or 48 h, the G0/G1 phase was increased by fludioxonil in a concentration-dependent manner. However, the S phase was decreased by fludioxonil (Figure 2A). At Ramos B cells which exposure of fludioxonil for 24 or 48 h, the S phase did not change with fludioxonil, but the G0/G1 phase was decreased by exposure of fludioxonil in a concentration-dependent manner ( Figure 2B).

Alteration of Cell Cycle-Related Genes Expression by Fludioxonil
To evaluate the effects of fludioxonil on the protein expression of cell cycle-related genes, such as cyclin D1 and cyclin E1, a Western blot assay was used using proteins which extracted from Jurkat T cells and Ramos B cells treated with fludioxonil (10 −7 -10 −5 M) for 24 or 48 h. In Jurkat T cells, protein expression of cyclin D1 and cyclin E1 was decreased by exposure to fludioxonil (10 −7 -10 −5 M) at 24 and 48 h compared to control, but there was significance at the highest concentration of fludioxonil ( Figure 3A). In Ramos B cells treated with fludioxonil (10 −5 M) for 48 h, protein expression of cyclin D1 was decreased, and cyclin E1 was decreased with exposure to fludioxonil (10 −7 -10 −5 M) at 24 and 48 h ( Figure 3B).   The protein expression of cell cycle-related genes, such as cyclin E1 and cyclin D1, in protein levels are shown as representative Western blot images. All bands were normalized by GAPDH and presented as % of controls. The value of the control (DMSO) was set as 1 and the values of the treatment groups were indicated as fold induction vs. control. The data are shown as the means ± standard deviation (SD) of three independent experiment. *p < 0.05 as compared with the control.

Apoptosis of Immune Cells by Fludioxonil Exposure
To confirm the effect of fludioxonil on apoptosis, Jurkat T cells and Ramos B cells following the treatment of fludioxonil were measured by FITC or AF488-annexin V/PI staining. Annexin V + /PIand Annexin V + /PI + sections show apoptosis. At 24 and 48 h, apoptosis of Jurkat T cells was increased by fludioxonil (10 −7 -10 −5 M) compared to control, but significance was present only at a high concentration of fludioxonil (10 −5 M) ( Figure 4A). In the Ramos B cells, apoptosis did not significantly increase at 24 h. However, at 48 h, apoptosis of Ramos B cells was increased by fludioxonil (10 −7 -10 −5 M), but significance was presented at only at a high concentration of fludioxonil (10 −5 M) ( Figure 4B). Apoptosis was increased in a time-and concentration-dependent manner in Jurkat T cells and Ramos B cells at 48 h, but at 24 h apoptosis was increased by fludioxonil in only Jurkat T cells. Thus, it was confirmed that fludioxonil can induce apoptosis in the lymphocytes, and Jurkat T cells are more sensitive as fludioxonil than Ramos B cells.

Decrease of Mitochondrial Membrane Potential by Fludioxonil
Fluorescence microscopy was used to assess the dysfunction of mitochondrial membrane potential in Jurkat T cells and Ramos B cells, as shown

Alteration of Apoptosis-Related Genes Expression by Fludioxonil
Western blot was accomplished to investigate whether the fludioxonil affects the protein expression of apoptosis-related genes, such as p53, Bcl-2-associated X protein (BAX), Bcl-2, and cleaved caspase-3, in Jurkat T cells and Ramos B cells or not ( Figure 6). The pro-apoptotic protein expression of p53, BAX, and cleaved caspase-3 were increased, but the anti-apoptotic protein expression of Bcl-2 was decreased by fludioxonil in Jurkat T cells treated with fludioxonil for 24 and 48 h. However, the quantified results present that cleaved caspase-3 and BAX was significantly increased, and Bcl-2 was significantly decreased ( Figure 6A). In the Ramos B cells treated with fludioxonil for 24 and 48 h, the protein expression of p53, BAX, and cleaved caspase-3 was increased and protein expression of Bcl-2 was decreased, at 24 h protein expression of cleaved caspase-3 was presented only in 10 −5 M fludioxonil. In the Ramos B cells treated with fludioxonil for 24 h, significance was presented in p53, cleaved caspase-3, and Bcl-2, and in the Ramos B cells treated with fludioxonil for 48 h, significance was presented in cleaved caspase-3 and BAX ( Figure 6B).

Increase of Fas Gene Expression by Fludioxonil
Fas is related with the extrinsic apoptotic pathway. Semi-quantitative RT-PCR was used to confirm the gene expression of Fas by the treatment of fludioxonil in Jurkat T cells and Ramos B cells (Figure 7). In Jurkat T cells treated with fludioxonil for 24 or 48 h, expression of Fas was increased by fludioxonil. Especially at 48 h, expression of Fas was more increased than the control ( Figure 7A), but in the Ramos B cells, fludioxonil did not alter the expression of the Fas gene ( Figure 7B).

304
Pesticides used for agricultural products increase the quality and protection from pests, insects, 305 fungi, and harmful weeds. Pesticides have properties of residue, and pesticide residues are easily 306 exposed to the environment and tend to be environmentally persistent [2]. These pesticide residues immune system disorders [3,4]. According to another study, atrazine, carbamate, and tributylin 309 were demonstrated to induce apoptosis, cell cycle arrest, and dysfunction in immune cells by 310 affecting the expression of apoptosis-related genes and cell cycle-related genes, and by decreasing 311 mitochondrial membrane potential [28][29][30][31]. In particular, fludioxonil was investigated in regard to 312 promoting oncogenesis [8,27,32]. Although fludioxonil has shown to induce oncogenesis, there was 313 no research in which fludioxonil affects the immune system. In particular, we employed Jurkat T

Discussion
Pesticides used for agricultural products increase the quality and protection from pests, insects, fungi, and harmful weeds. Pesticides have properties of residue, and pesticide residues are easily exposed to the environment and tend to be environmentally persistent [2]. These pesticide residues cause many diseases and health risks, such as cancer, disrupting the nervous system, allergies, and immune system disorders [3,4]. According to another study, atrazine, carbamate, and tributylin were demonstrated to induce apoptosis, cell cycle arrest, and dysfunction in immune cells by affecting the expression of apoptosis-related genes and cell cycle-related genes, and by decreasing mitochondrial membrane potential [28][29][30][31]. In particular, fludioxonil was investigated in regard to promoting oncogenesis [8,27,32]. Although fludioxonil has shown to induce oncogenesis, there was no research in which fludioxonil affects the immune system. In particular, we employed Jurkat T cells and Ramos B cells because we considered that T cells and B cells were basic immune cells in adaptive immunity.  [33], and are derived from hematopoietic stem cells, but have distinguished roles in immune response. T cells are involved in cellular immunity and B cells play a role in the humoral immunity. T cells activate B cells, macrophages, and cytotoxic T cells, and kill the infected cells and tumor cells. B cells secrete the antibodies that eliminate the antigens [34,35]. Furthermore, fludioxonil did not affect the cell viability of normal cells, such as the human lung diploid fibroblast cell line WI-26. These results suggest that fludioxonil decreased the cell viability in immune cells but did not affect normal cells.
In addition, fludioxonil induced cell cycle arrest. Cell cycle arrest by fludioxonil was confirmed using flow cytometry by PI staining. Then, Western blot assay proved that fludioxonil modulated the protein expression of cell cycle-related genes, such as cyclin E1 and cyclin D1. These proteins are related with cell cycle G1 and S phase and critical kinases in cell cycle regulation [36]. Cyclin D1 applies in the G1 phase and cyclin E1 plays a role in the cell cycle G1/S transition [37,38]. In Jurkat T cells, the G0/G1 phase was increased and the S phase was decreased by fludioxonil, and protein expression of cyclin E1 and cyclin D1 was decreased by fludioxonil at 24 and 48 h. In the Ramos B cells, fludioxonil decreased the G0/G1 phase and decreased the protein expression of cyclin D1 at 24 and 48 h, but the protein expression of cyclin E1 did not change at 24 h. The decrease of cyclin E1 implies that the G0/G1 phase does not progress to the S phase, and the decrease of cyclin D1 implies that the G0/G1 phase was decreased. In Jurkat T cells, G1 cell cycle arrest was induced by fludioxonil [39]. In cell cycle analysis, the change at 24 h in Ramos B cells was not linked to the expression of cyclins. However, both cyclin E1 and cyclin D1 were decreased by fludioxonil in Jukat T cells. Therefore, fludioxonil has effects on the cell cycle via alteration of cell cycle regulation proteins, such as cyclins. Cell cycle arrest was induced by fludioxonil in human Jurkat T cells and Ramos B cells.
The apoptosis by fludioxonil in Jurkat T cells and Ramos B cells were confirmed by decreasing mitochondrial membrane potential, analysis of annexin V/PI staining, and altered protein expression of apoptosis-related genes and increased gene expression of Fas/Fas ligand. In Jurkat T cells, early apoptosis (Annexin V + /PI -), late apoptosis (Annexin V + /PI + ), and green fluorescence related with mitochondrial membrane potential loss were increased by treatment with fludioxonil for 24 and 48 h. In Ramos B cells, early apoptosis, late apoptosis, and green fluorescence were increased by treatment with fludioxonil for 48 h. Annexin V is phospholipid-binding protein that binds to phosphatidylserine (PS). PS is exposed at the cell surface which underwent apoptosis and recognized the apoptotic cells [40]. The dysfunction of mitochondrial membrane potential leads to apoptosis. The mitochondrial membrane potential was measured by JC-1, and JC-1 dye exhibits two fluorescences: one is red fluorescence, reflecting higher membrane potential, and the other is green fluorescence, indicating dysfunction of membrane potential [41,42]. Proteins related with apoptosis were confirmed by Western blot assay. p53, BAX, and cleaved caspase-3 were increased in Jurkat T cells that were treated with fludioxonil for 24 and 48 h, but Bcl-2 was decreased in Jurkat T cells that were exposed to fludioxonil for 24 and 48 h. In Ramos B cells, protein expression of p53, BAX, Bcl-2, and cleaved caspase-3 was altered with the exposure to fludioxonil for 24 and 48 h. p53 can induce the apoptotic pathway in the cell, and p53 up-regulated BAX, which is a pro-apoptotic protein, and down-regulated Bcl-2, which is an anti-apoptotic protein [43,44]. These proteins activated caspase-3 to cleaved caspase-3, and cleaved caspase-3 induces apoptosis [45].
These proteins are related to the intrinsic apoptotic pathway, and the intrinsic apoptotic pathway occurs through the mitochondria [46]. The other apoptosis pathway is the extrinsic apoptotic pathway, which is related with the Fas/Fas ligand. Fas and Fas ligand induce apoptosis through activating caspase-3 [47]. Fas gene expression was confirmed. In Jurkat T cells, Fas was increased by fludioxonil treatment for 24 and 48 h. However, in Ramos B cells treated with fludioxonil for 24 and 48 h, fludioxonil did not change Fas. As a result, fludioxonil induces apoptosis via the increase in pro-apoptotic-related proteins, Fas gene, a decrease in anti-apoptotic-related protein, and induction of dysfunction of mitochondrial membrane potential. Thus, these results present that fludioxonil may induce intrinsic and extrinsic apoptotic pathways and dysfunction of mitochondrial membrane potential in human immune cells, Jurkat T cells, and Ramos B cells.

Conclusions
In conclusion, it was found that fludioxonil may induce apoptosis by inhibiting cell proliferation through cell cycle arrest. Apoptosis was induced by the intrinsic apoptotic pathway mediated in mitochondria and the extrinsic apoptotic pathway and dysfunction of mitochondrial membrane potential. In addition, Jurkat T cells appeared to be more sensitive than Ramos B cells in this study. Therefore, as shown in Figure 8, the present study indicates that fludioxonil pesticides can induce cytotoxicity in human immune cells. That is, fludioxonil might cause of various immunological diseases by cytotoxicity. Figure 8. Cytotoxicity of fludioxonil on human immune cells. Fludioxonil leads to apoptosis that is mediated by the mitochondria and death receptor (Fas). In mitochondria, fludioxonil depolarizes membrane potential and increases expression of p53, then p53 alters the expression of BAX and Bcl-2. Increased BAX activates cleaved caspase-3. Fludioxonil increases gene expression of Fas, which activates cleaved caspase-3. Activated cleaved caspase-3 induces apoptosis. Additionally, fludioxonil decreases the expression of cell cycle-related genes, such as cyclin D1 and E1, and decreased cyclin induces cell cycle arrest.

Conflicts of Interest:
The authors do not have any conflicts of interest to declare.