The insecticide chlorpyrifos leads to genotoxic effects by triggering oxidative stress and apoptosis in Sertoli cells

Abstract

Chlorpyrifos (CPF) is an insecticide that humans are exposed to when used for agricultural and domestic purposes. Many studies have shown that CPF has adverse effects on human health and causes toxicity in various cells and tissues. Since there are few studies in the literature examining the effects of CPF on the male reproductive system, Sertoli cells, which are crucial to the continuation of spermatogenesis, were chosen as a model in our study. The objective of this study was to investigate the cytotoxic, oxidative stress–related, apoptotic, and genotoxic effects of chlorpyrifos in TM4 Sertoli cells. In this study, Sertoli cells were exposed to two concentrations of CPF (100 and 500 μM) for 24 h. The cytotoxic and genotoxic potential of CPF in Sertoli cells was evaluated by measuring metabolic activity, malondialdehyde content, antioxidant enzyme levels, apoptosis rates, and DNA damage. According to the findings, applied CPF concentrations increased oxidative stress and decreased antioxidant enzyme levels in Sertoli cells. The results indicated that cell viability and the nuclear division index decreased in a concentration-dependent manner, while apoptotic cells, the micronucleus, and comet formation increased. Consequently, the results suggest that DNA damage and apoptosis play a major role in the mechanisms underlying the cytotoxic effects of CPF in Sertoli cells, establishing CPF as a strong genotoxic agent.

Keywords

Chlorpyrifos Sertoli cells oxidative stress DNA damage apoptosis micronucleus assay

Introduction

Pesticides are chemicals that are widely used in agricultural production and settlements to control harmful organisms such as insects, rodents, fungi, and weeds and to reduce their negative effects (Kaushal et al., 2021). Although the benefits of pesticides in preventing agricultural product loss are known, if not applied in correct and appropriate doses, pesticide groups can leave residue on foods and pose a risk to human health. Organophosphate compounds in the pesticide group are widely used worldwide owing to their high efficiency and low half-life (Huang et al., 2021).

Chlorpyrifos (CPF) is a chemical compound that belongs to the group of insecticides classified as organophosphates (Huang et al., 2021). After being granted a patent by the Dow Chemical Corporation in 1966, CPF’s uses expanded to include households and agriculture (Hites, 2021). CPF has been found to accumulate on surfaces such as pillows, carpets, and soft toys after application, even if applied according to the manufacturer’s instructions (Gurunathan et al., 1998). CPF, a substance with numerous uses that protects against damage from insects and arthropods, affects the nervous system of insects by inhibiting the acetylcholinesterase enzyme (Chen et al., 2018). CPF is being used more frequently since it is safer than parathion and other parathion-related substances. The World Health Organization has classified CPF to be moderately harmful to humans due to its acute toxicity (Huang et al., 2021). By conducting a considerable investigation, the Environmental Protection Agency concluded in 2011 that humans were mostly exposed to CPF through the residue it left on food (Ozturk et al., 2022). CPF can also be ingested through drinking water sources or residues in food processing industries.

The tolerable daily intake for exposure to CPF determined by the European Food Safety Authority is 0.1 µg/kg/day (Kopjar et al., 2018). After CPF enters the body, it is converted to CPF-oxone by CYP450 enzymes and is rapidly hydrolyzed to 3,5,6-trichloro-2-pyridinol. Approximately 70% of the exposed dose of CPF is excreted in the urine as 3,5,6-trichloro-2-pyridinol (Badr, 2020). According to studies, the half-life of CPF in soil can range from 60 to 120 days, and these values depend on the composition of the soil. Moreover, it has been determined that the half-life of CPF in water is between 35 and 78 days (Huang et al., 2021).

Exposure to CPF has been found to cause liver dysfunction, immunological abnormalities, toxicity in developing organisms, genotoxicity, cytotoxicity, and alterations in neurochemical and behavioral functions (Kaushal et al., 2021). Moreover, studies on the effects of low doses of CPF on zebrafish reproductive and amphibian brain development have been conducted (Yu et al., 2015). New studies on CPF have revealed that it affects the intestines, which results in an increase in obesity and insulin resistance (Liang et al., 2019). CPF can interact with the phospholipids in plasma membranes because of its structural hydrophobicity, which results in lipid peroxidation. It was shown in a detailed look into the possible toxicity of CPF that malondialdehyde (MDA) levels were elevated and superoxide dismutase (SOD) activity dropped in various tissues of rats exposed to the chemical (Alipanah et al., 2022). CPF can bind to DNA, causing chromosomal abnormalities and an increase in the frequency of micronuclei production, according to numerous studies (Jamil et al., 2005; Li et al., 2015).

In a study to examine the effects of CPF on the male reproductive system, it was found that infertility occurred, and sperm motility and density were reduced in mice administered intragastric doses of CPF (Zhang et al., 2020). In another study, different CPF concentrations were treated in mouse spermatogonia, Sertoli, and Leydig cells to assess the cytotoxic and genotoxic effects on the male reproductive system. According to the findings, CPF dose-dependently promotes apoptosis and decreases cell growth (Chen et al., 2018). A study using CPF-containing insecticides found that exposure to the chemicals lowered the levels of the hormones testosterone, luteinizing hormone, follicle stimulating hormone, SOD, and glutathione (GSH) in the testes of rats (Abdel-Razik et al., 2021).

Studies on the detrimental effects of CPF on many tissues, organs, and cells in living organisms exist, but there are very few on the male reproductive system, particularly Sertoli cells. Unlike the study by Chen et al. (2018), which examined multiple cell types and mainly focused on apoptosis with a limited set of genotoxicity assays, the present study focused exclusively on TM4 Sertoli cells and evaluated a broader spectrum of endpoints, including cytotoxicity, oxidative stress parameters, apoptosis, and multiple genotoxicity assays, along with cell proliferation indices. In this study, we focused on cytotoxicity, oxidative stress, apoptosis and various genotoxicity tests in order to determine the genotoxic effects of CPF in Sertoli cells, which are responsible for the continuity of spermatogenesis in the male reproductive system.

Materials and methods

Chemicals and reagents

CPF (98% purity, Cat. no. 45395), agarose low-melting-temperature (LMTA), agarose normal-melting-temperature (NMTA), cytochalasin B, 4′,6-diamidino-2-phenylindole (DAPI), dimethyl sulfoxide (DMSO), Giemsa, propidium iodide, and Hoechst 33342 were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modified Eagle medium/Ham’s F-12 medium (DMEM/F12), fetal bovine serum (FBS), horse serum (HS), penicillin-streptomycin solution, 0.05% trypsin-EDTA were obtained from Wisent Bioproducts (St. Bruno, Quebec, Canada). Other chemicals and reagents were all analytical grade and purchased from Biomatik Company (Ontario, Canada).

Cells, culture conditions and treatments

TM4 Sertoli cells (ATCC® CRL-1715) used in all experiments (Passages 11–25) were cultured in medium supplemented with 5% HS, 2.5% FBS, and 1% penicillin-streptomycin solution. Sertoli cells were maintained in a humidified incubator at 37°C with 5% CO₂ and 95% relative humidity. Cells were grown and allowed to adhere overnight before CPF treatment. Subsequently, cells were exposed to CPF at different concentrations (0–500 µM) and incubated for 24 h at 37°C in a humidified incubator with 5% CO₂. The control cells were grown in complete medium. Each of the three independent experiments was performed in triplicate. Stock solutions of CPF were prepared in DMSO and diluted with culture medium to the desired concentrations. The final concentration of DMSO in all treatment and control groups was 0.05% (v/v) to avoid solvent-induced cytotoxicity. The CPF concentrations selected for all subsequent experiments were 100 and 500 µM and, according to cell viability results, these concentrations had no effect on cell viability (90–95%) and reduced cell viability to approximately 60%, respectively. Furthermore, these concentrations were chosen based on our preliminary cytotoxicity results and previous literature in which CPF was used in the range of 10–1000 µM in various cell types, including Sertoli cells (Chen et al., 2018; Gao et al., 2021; Zhang et al., 2020).

Cell viability by MTT assay

The cytotoxic effects of CPF on TM4 Sertoli cells were assessed using a colorimetric technique called the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test (Roche, Germany). TM4 cells were seeded in 96-well culture plates at a density of 5 × 10³ cells per well and incubated overnight for MTT cell viability tests. The cells were then incubated for 24 h at 37°C with 5% CO₂ after being exposed to two different concentrations of CPF, 100 µM and 500 µM. Following the completion of the CPF exposure, 10 μL of MTT I solution was added to each well, and it was then incubated for 4 h at 37°C in a CO₂ incubator. Crystals of water-insoluble formazan are produced after this incubation. In order to dissolve the formazan crystals produced by live cells, 100 μL of MTT II solution (SDS) was applied to each well. Thereafter, each well was placed in a CO₂ incubator overnight. The optical densities of the cells were evaluated at 540 nm at the completion of this time using an enzyme-linked immunosorbent assay (ELISA). The viability of control cells which were not exposed to the test chemical was assumed to be 100%. The percentage of viable cells was calculated using the following formula:

Cell viability = (Absorbance of treated cells / Absorbance of control cells) \times 100

Preparation of samples for biochemical analysis

For biochemical analysis, 1 × 10⁶ TM4 cells were seeded in six-well culture dishes and allowed to adhere overnight. The cells were exposed to CPF concentrations for 24 h. After exposure, cells were harvested with trypsin and placed into an ice-cold lysis buffer (Tris-HCl pH 7.2). Afterward, the cells were sonicated with an ultrasonicator to break the cell membranes and reveal the cell composition. The acquired cell suspension was centrifuged at 14000×g in a Nüve brand refrigerated centrifuge, and the supernatant was collected. The supernatants taken were brought to −86°C to be used in lipid peroxidation and antioxidant enzyme analyses.

Determination of lipid peroxidation

MDA production was measured to assess lipid peroxidation using the Devasagayam and Tarachand method (Devasagayam and Tarachand, 1987). This technique was based on the thiobarbituric acid reaction in boiling water at an acidic pH. Cell extract was added to a reaction mixture that contained 10 mM KH₂PO₄ and 0.15 M of Tris-HCl buffer (pH 7.4). The tubes were then stopped with 10% trichloroacetic acid and thiobarbituric acid after being incubated for 20 min at 37°C in a shaking bath. After that, the tubes stayed 20 min immersed in a bath of boiling water. After cooling, a spectrophotometer was used to detect the absorbance at 532 nm.

Determination of antioxidant system parameters

SOD enzyme activity was demonstrated using the Marklund and Marklund method, which was based on the prevention of auto-oxidation of pyrogallol at alkaline pH by the SOD enzyme (Marklund and Marklund, 1974). The amount of enzyme required to reduce the auto-oxidation of pyrogallol by 50% is defined as one unit of SOD activity. The catalase (CAT) activity was shown using the Sinha procedure (1972). H₂O₂, a reactive oxygen species that damages cells, is decomposed into water and molecular oxygen by the CAT enzyme. The principle of this procedure is that the dark blue-violet color created by dichromate in acetic acid in the presence of hydrogen peroxide is reduced to chromic acetate as a result of heating, and the color changes to bright green. This pale green color gives an absorbance at a wavelength of 570 nm. To protect organisms from oxidative damage, glutathione peroxidase (GPx) detoxifies peroxides within the cell. In the presence of reduced GSH, the GPx enzyme catalyzes the process that decomposes hydrogen peroxide into oxidized GSH and water. The Hafeman technique was used to assess the amounts of the GPx enzyme (Hafeman et al., 1974). The experiment is based on the spectrophotometric detection of the molecule generated by the reaction of GSH consumed by the GPx enzyme with the 5,5′-dithio-bis (2-nitrobenzoic acid) reagent at 412 nm.

Apoptosis analysis by double fluorescent staining

After CPF exposure, double staining was conducted immediately on live cells, without fixation. Propidium iodide and bis-benzimidazole Hoechst 33342 were both added to the culture medium at a final concentration of 1 µg/mL for each reagent before being cultured for 15 min at 37°C in the dark. Fluorescence microscopy (Olympus IX71, Tokyo, Japan) was used to monitor the cells using two filters (DAPI fluorescent filter, excitation 340–380 nm; and rhodamine filter, excitation 530–560 nm) and photographed in series with equal intervals by the Olympus DP72 (Tokyo, Japan) video camera. The ratio of viable, apoptotic, and dead cells was calculated via a total of 1000 cells counted for each experimental group in a series of photographs.

Genotoxicity by single-cell gel/comet assay

The comet test was used to detect DNA damage at the level of a single eukaryotic cell. The comet test is based on the principle that DNA isolated from living tissues is fixed in an agarose gel and electrophoretically executed, resulting in a comet image by migrating at different speeds due to the different electrical charges and molecular weights of the damaged DNA fragments. In accordance with the Singh et al. (1988) method, comet experiments were performed in an alkaline environment. In this method, TM4 Sertoli cells were seeded in 24-well plates in a concentration of 1 × 10⁵ cells/mL and treated for 24 h with two different CPF concentrations and H₂O₂ as the positive control. After exposure, Sertoli cells were harvested, and cell suspensions (20 µL) were resuspended in 120 µL of low-melting point agarose and then dispersed on slides covered with 1.5% normal-melting point agarose. Following agarose solidification at 4°C for 1 h, the slides were immersed in freshly prepared and pre-chilled lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH: 10; 10% DMSO and 1% Triton-x 100) in the dark at 4°C for 1 h. Then, the slides were transferred to a horizontal electrophoresis tank and treated with freshly prepared chilled alkaline unwinding solution (300 mM NaOH and 1 mM EDTA, pH > 13) to denature the DNA at 4°C for 20 min. Electrophoresis was performed with a precooled alkaline electrophoresis solution at 25 V and 300 mA for 30 min. After electrophoresis was complete, the slides were washed with distilled water, treated with neutralization buffer (0.4 M Tris, pH: 7.5) three times for 5-min periods, and fixed with absolute ethanol for 10 min. Subsequently, the slides were stained with 4′,6-diamidino-2-phenylindole (2 µg/mL) and comets were scored using an Olympus IX71 inverted fluorescent microscope. For each group, 100 randomly selected nucleoids were visually scored to determine damage after being divided into distinct groups depending on the length and form of their tails, and the damage was determined using the CometScore 2.0 software.

Cytokinesis-blocked micronucleus (CBMN) assay

This assay was performed according to Fenech with some modifications (Fenech, 2007). TM4 Sertoli cells were cultured in 6-well plates with 1.5 × 10⁵ cells/mL and left overnight to adhere. The cells were treated with 100 µM and 500 µM CPF for 24 h. Following the exposure period, the experimental medium was removed, and the cells were incubated with a medium containing cytochalasin B (final concentration 4 µg/mL) for 20 h. The cells were then treated with trypsin-EDTA for harvesting after being exposed to cytochalasin B. Immediately upon harvesting, the cells were centrifuged, the supernatant was removed, and the cells were then suspended in one drop of 1% formaldehyde-added hypotonic solution (0.075 M KCl) and incubated for 5 min. Cell suspensions were centrifuged once more after being fixed using Cornay’s reagent (3:1 methanol/glacial acetic acid). Cells were centrifuged, resuspended in Cornay’s reagent, and then prepared by dropping the cell solution and drying them by air on slides. Slides were stained for 8 min with 4% Giemsa in phosphate buffer (Na₂HPO₄ 0.06 M and KH₂PO₄ 0.06 M, pH 6.8) before being rinsed with water. A brightfield microscope (Olympus IX71) was used to score from each slide to ascertain the frequency of micronucleus (MN), nucleoplasmic bridges (NPBs), and/or nuclear buds (NBUDs). The microscopic slides were analyzed by two independent scorers in a blinded, random manner. Binucleated cells with micronuclei (BNMN) were quantified in at least 1000 cells/concentration, including mono-, bi-, tri-, and tetra-nucleated cells. Cytokinesis-blocked proliferation index (CBPI) was calculated by counting 1000 cells/concentration using the following equation (1) (Lorge et al., 2008):

CBPI = \frac{(M o n o N c) + (2 x B i N c) + (3 x M u l t i N c)}{T o t a l N o . o f c e l l s},

(1)

where “MonoNc” represents mono-nucleated cells, “BiNc” represents bi-nucleated cells, and “MultiNc” represents multi-nucleated cells (tri- and tetra-nucleated cells).

The replicative index (RI), a measure of cell division kinetics, was computed by counting the percentage of cells per individual that had one, two, three, or more nuclei and scoring at least 500 cells per concentration or sample. RI was evaluated using equation (2) (Lorge et al., 2008):

R I = \frac{(((1 x B i N c) + (2 x M u l t i N c)) + (T o t a l N o . o f c e l l s)) t e s t}{(((1 x B i N c) + (2 x M u l t i N c)) + (T o t a l N o . o f c e l l s)) c o n t r o l} \times 100,

(2)

The cytostasis percentage provides data on cell kinetics and was estimated using formula (3) (Lorge et al., 2008):

% cytostasis = 100 - 100 \times {\frac{(C B P I t e s t - 1)}{(C B P I c o n t r o l - 1)}}

(3)

where “CBPI test” represents the mean CBPI of each concentration used in the experiment and “CBPI control” represents the mean CBPI of the control.

In addition, the nuclear division index (NDI) was assessed in at least 500 cells/concentration using the formula below:

N D I = \frac{(1 x M o n o N c) + (2 x B i N c) + (3 x T r i N c) + (4 x T e t r a N c)}{(T o t a l N o . o f c e l l s)},

where “TriNc” represents tri-nucleated cells and “TetraNc” represents tetra-nucleated cells.

Statistical analysis

The fundamental parameters were acquired using descriptive statistics, absolute and relative frequencies, and additional analyses were calculated using GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). In all tests, statistical significance was set at p < 0.05. The Shapiro-Wilk test was used to determine the normality of the residues, and the results were found to be normally distributed. The variances’ homogeneity was evaluated using Bartlett’s test, and the variances were found to be homogeneous. Consequently, one-way analysis of variance with Tukey’s multiple comparison test was used for all analyses of this experiment. The results were expressed as means ± standard errors of the mean.

Results

Cytotoxicity results

The MTT assay was used to evaluate the cell viability of TM4 Sertoli cells induced by CPF at exposure concentrations ranging from 0 to 500 μM for 24 h. The viability of Sertoli cells at 400 and 500 µM CPF concentrations was recorded as 74.49% and 59.13%, respectively (Figure 1). Figure 2 shows morphological modifications in Sertoli cells following exposure to CPF concentrations of 100 and 500 μM in comparison to the control group, particularly at the applied concentration of 500 μM.

Measurement of lipid peroxidation and antioxidant enzymes

According to the findings of this study, CPF exposure had a toxic effect on Sertoli cells and significantly increased the amount of lipid peroxidation at CPF concentrations of 100 and 500 μM (Figure 3(A)). Figures 3(B) and (C) display the calculated SOD, CAT, and GPx enzyme levels in the CPF-treated groups of TM4 Sertoli cells after the 24 h experiment. SOD (Figure 3(B)) and CAT (Figure 3(C)) enzyme levels were evaluated between the control group and both CPF concentrations, and both groups showed a statistically significant decline (p < 0.001). There was a significant decrease in GPx enzyme activity after exposure to 100 μM CPF (p < 0.05) and 500 μM (p < 0.01) (Figure 3(D)).

Apoptosis results

The percentages (Table 1) and images (Figure 4) of viable, apoptotic, and dead cells in Sertoli cells following CPF exposure were determined by double fluorescence staining. CPF treatment resulted in a significant decrease in the number of viable cells in Sertoli cells. The apoptotic cell rate increased considerably at all CPF concentrations. Whereas the dead cell proportion substantially increased only 500 μM CPF-treated group in Sertoli cells.

Table 1.

Concentration-dependent effects of CPF on viable, apoptotic, and dead cells in Sertoli cells.

	Control	100 µM CPF	500 µM CPF
Viable cells (%)	99.4 ± 1.4	82.1 ± 1.7^*	66.8 ± 1.1^*
Apoptotic cells (%)	0.5 ± 0.0	17.4 ± 0.9^*	24.0 ± 1.4^*
Dead cells (%)	0.1 ± 0.0	0.5 ± 0.0	9.2 ± 0.8^*

Results from three different experiments with triplicate measurements were presented with a mean standard error. Significance at p < 0.05. *Significant p-value versus the control group. CPF: chlorpyrifos.

Alkaline comet assay results

Figure 5 displays Sertoli cells damaged following exposure to CPF at 100 μM and 500 μM concentrations, damaged Sertoli cells exposed to 150 μM H₂O₂ as a positive control, and undamaged Sertoli cells exposed to DMSO (0.05%) alone as a negative control. Comparing the results of the comet assay in Sertoli cells exposed to different CPF concentrations for 24 h, there was a significant increase in both the 100 μM applied concentration (6x increase, p < 0.01) and the 500 μM concentration (9x increase, p < 0.001) in the tail length compared to the negative control (Table 2). The average DNA in the tail (%) elevated at concentrations of 100 μM CPF (1.7x increase, p < 0.01) and 500 μM CPF (3.5x increase, p < 0.001) when compared with the negative control, as shown in Table 2. In comparison to the negative control, there were also noticeable increases in the olive tail moment, one of the parameters of the comet assay, in the samples treated with CPF. When compared to negative control, it was determined that the applied CPF concentration increased to 1.9-fold (p < 0.05) at the 100 μM concentration and up to 2.9-fold (p < 0.01) at the 500 μM concentration.

Table 2.

DNA damage of Sertoli cells after exposure to CPF in comet assay.

	Control	100 µM CPF	500 µM CPF	Positive control (150 µM H₂O₂)
Tail length	6.4 ± 0.7	38.2 ± 2.4^*	58.7 ± 5.2^*	61.5 ± 5.8^*
Tail DNA (%)	8.5 ± 1.1	14.6 ± 1.2^*	30.0 ± 3.1^*	49.3 ± 2.6^*
Tail olive moment	2.4 ± 0.2	4.5 ± 0.3	7.1 ± 0.7^*	8.5 ± 1.2^*

Cytokinesis-block micronucleus assay results

The effects of CPF on CBPI, RI, NDI, and cytostatic alterations in TM4 cells were compared to the control group (Figures 6(A)–(D)). When the CBPI values, an indication of cytotoxicity, were analyzed, a substantial decrease in the applied 500 μM concentration of CPF was detected (Figure 6(A), p < 0.01). It was found that CPF influenced cell division and resulted in 2.5% and 16% cytostasis at concentrations of 100 and 500 μM, respectively (Figure 6(B), p < 0.05 and p < 0.001). This was estimated after considering the findings of cytostasis generated by hazardous chemicals. RI values indicating differences in cellular toxicity demonstrated an effective reduction at 500 μM CPF concentration (Figure 6(C), p < 0.001). The data analysis revealed that, despite the decrease in NDI observed in the group exposed to 500 μM CPF, there was no noticeable difference between the control group and the 100 μM concentration. (Figure 6(D), p < 0.001). Table 3 displays the findings of MN, NPBs, NBUD, and NBLEB identified in binucleate cells following CPF exposure in Sertoli cells. When the data were analyzed, it was shown that a 100 μM concentration of CPF caused no DNA damage in Sertoli cells; however, a 500 μM concentration induced chromosomal alterations (clastogenicity/aneugenicity) by increasing the frequency of MN, NBP, NBUD, and NBLEB (p < 0.05).

Table 3.

Incidence of MN, NPB, NBUDs, and NBLEBs as nuclear abnormalities in Sertoli cells exposed to different concentrations of CPF.

		Control	100 µM CPF	500 µM CPF	Positive control (150 µM H₂O₂)
Binucleated cells (%)	MN	0.0 ± 0.0	1.9 ± 0.5	3.8 ± 0.5^*	48.7 ± 1.9^*
	NPB	0.9 ± 0.1	2.9 ± 0.7	38.7 ± 2.4^*	43.6 ± 2.1^*
	NBUDs	1.3 ± 0.3	0.9 ± 0.1	7.2 ± 1.6^*	12.1 ± 1.3^*
	NBLEBs	1.1 ± 0.2	1.3 ± 0.3	3.5 ± 0.9^*	5.9 ± 0.9^*

Results from three different experiments with triplicate measurements were presented with a mean standard error. Significance at p < 0.05. *Significant p-value versus the control group. CPF: chlorpyrifos, MN: micronucleus, NPB: nucleoplasmic bridge, NBUDs: nuclear buds, and NBLEBs: nuclear blebs.

Discussion

Several studies have been carried out recently to assess the risk and toxicity of CPF on the male reproductive system (Albasher et al., 2020; Zhang et al., 2020). According to reported research, CPF can cause alterations to sperm motility and a reduction in sperm count (Babazadeh and Najafi, 2017). Despite studies, the mechanism of CPF toxicity in the male reproductive system has not been elucidated in the literature. In the present study, the toxicity of CPF in Sertoli cells, one of the most important cells of the male reproductive system, was evaluated by examining the changes in cytotoxicity, oxidative stress, genotoxicity, and apoptosis parameters.

The effect of CPF on cell viability has been the subject of several studies. According to the results of the MTT test, 12 µg/mL CPF exposure resulted in a significantly lower cell viability in the CPF-induced group than in the control group in human lymphocytes (Navaei-Nigjeh et al., 2015). In another study, the HepG2 cell line was exposed to concentrations of CPF of 400, 556.63, 774.6, 1077.91, and 1500 µM. According to the findings, cell viability reduced in a concentration-dependent manner (Zhou and Li, 2018). In another investigation, nerve cells were exposed to 0.1, 1, 10, and 100 µM CPF, and the experiment revealed that nerve cell viability reduced in parallel with administered concentrations (Giordano et al., 2007). In different studies with Leydig, Sertoli, and spermatogonium cells, CPF (in the range of 10–1000 µM) was found to suppress cell viability (Chen et al., 2018; Gao et al., 2021; Zhang et al., 2020). Similar to the studies in the literature, it was revealed that CPF inhibited cell viability and caused morphological damage to the cells at a concentration of 500 µM in Sertoli cells.

Lipid peroxidation induced by damage to normal cell function as a result of CPF exposure is an indicator of oxidative stress. CPF treatment to MCF-7 and MDA-MB-231 cells at concentrations of 0.01, 1, 10, and 100 µM resulted in a concentration-dependent increase in lipid peroxidation (Moyano et al., 2021). Increased levels of lipid peroxidation were induced in neurons by a 100 µM concentration of CPF (Lee et al., 2014). Human lymphocytes were used in research, and lymphocytes treated with 0, 100, 500, 1000, and 2000 ppm CPF had elevated MDA levels (Deeba et al., 2017). It was discovered that 100 µM CPF caused a substantial increase in MDA level in the study conducted with PC12 cells (Lee et al., 2012). It was discovered that CPF promoted lipid peroxidation in Sertoli cells in accordance with in vitro studies.

The increase in reactive oxygen species, which causes oxidative stress, leads to the consumption of antioxidant enzymes and negatively affects antioxidant homeostasis. An analysis of human umbilical cord blood erythrocytes induced with 4, 40, and 400 nM concentrations of CPF revealed a concentration-dependent decrease in SOD and CAT levels (Quintana et al., 2018). SOD and GSH levels in the testis were significantly reduced in a different study after rats received a 9 mg/kg dose of CPF (Hassan et al., 2021). In their investigation using rat testis tissue, Alipanah et al. (2022) found that treatment with doses of 3.25, 6.75, and 3.5 mg/kg of CPF significantly reduced the levels of CAT and SOD compared with the control group. The reduction in the activities of SOD, CAT, and GPx enzymes in Sertoli cells, as well as studies assessing the antioxidant system parameters of CPF in rat testis tissue, were concluded to disrupt the oxidant/antioxidant balance of CPF at the cellular level (Albasher et al., 2020; Owumi et al., 2021). The major antioxidant enzymes in Sertoli cells, SOD, CAT, and GPx, were measured in this study, and it was shown that CPF causes a malfunction in the antioxidant system. It was discovered for the first time that CPF interferes with the activity of enzymes that maintain antioxidant/oxidant equilibrium in Sertoli cells, supporting research with various cells and tissues.

Apoptosis is a highly complex and dynamic process that is involved in physiological processes and is necessary for the survival of living organisms (Delhalle et al., 2003). Exposure to environmental pollutants and chemicals induces apoptosis in biological systems by causing toxicity (Franco et al., 2009). CPF may induce apoptosis in cells and organs, according to recent studies (Albasher et al., 2020; Li et al., 2009; Lin et al., 2023). In a previous study, Sertoli and spermatogonium cells were exposed to 10, 25, and 50 µM concentrations of CPF, and it was demonstrated that apoptosis was triggered in the cells (Chen et al., 2018). In a different investigation, CPF was applied to cultured murine neuroblastoma cells at concentrations between 50 and 300 µM. The findings of the study revealed that apoptosis was in fact caused in cells depending on concentration (Lin et al., 2023). In another study with cultured human T cells, 0–100 ppm concentrations of CPF were applied to the cells. According to the results, caspase-3 activity was responsible for inducing apoptosis in cells (Li et al., 2009). Li et al. (2015) reported that CPF enhanced apoptotic rates in the range of 90–400 µM utilizing the Annexin V technique in their investigation employing several cell lines. When apoptosis rates were examined in our study using the double-fluorescent labeling technique, it was shown that CPF significantly induced apoptosis in Sertoli cells.

In studies reported in recent years, it has been shown that CPF causes genotoxicity by affecting chromosomal DNA in cells. It was demonstrated in research by Patnaik and Padhy (2016) that CPF administration to HepG2 cells in the concentration range of 5–70 mg/L enhanced the development of micronuclei and the length of comet tails in cells. In another study evaluating the genotoxic potential of CPF in bovine lymphocyte cells, CPF was treated to the cells in a concentration range of 5.9–94.0 μg/mL. In conclusion, it has been noted that, depending on the concentration, CPF increases the frequency of nuclear bud and micronucleus formation in cells containing binucleate (Ferre et al., 2020). A previous study found a dose-dependent increase in micronucleus frequency in bone marrow cells (3.1 and 6.2 mg/kg CPF) isolated from rats and an increase in comet tail length in blood cells (6.2 mg/kg CPF) (Ezzi et al., 2016). Human peripheral blood cells were demonstrated to produce genotoxicity by lengthening the comet tail at various concentrations (0.1, 0.2, 0.5, and 1.0 mg/mL) in research examining CPF-induced DNA damage (Jamil et al., 2005). It was discovered that CPF triggered comet formation in the concentration ranges of 200–500 µM and 90–250 µM, respectively, in a work using HeLa and kidney cells (Li et al., 2015). In our study investigating the DNA damage caused by CPF in Sertoli cells by more than one mechanism, it was revealed that 100 and 500 µM CPF concentrations cause genotoxicity by disrupting chromosome instability, creating DNA breaks, and showing clastogenic effects.

This study aimed to clarify the fundamental mechanism of action of chlorpyrifos through oxidative stress, apoptosis, and genotoxicity; however, it should also be acknowledged that the study had limitations. More detailed studies can be conducted to examine the apoptotic changes that occur in Sertoli cells due to chlorpyrifos exposure at the gene and protein levels. It should not be ignored that in vitro studies alone may be insufficient, and in vivo studies are needed to elucidate the mechanism of action on humans. Furthermore, it should be noted that the CPF concentrations used in this study, particularly 500 µM, are substantially higher than the expected blood concentrations in vivo and may represent supra-physiological exposure levels. While such concentrations are frequently employed in in vitro toxicology studies to explore mechanistic pathways, caution should be exercised when extrapolating these findings to in vivo scenarios. The effects observed exclusively at 500 µM may not occur at environmentally or physiologically relevant exposure levels in humans; however, these results provide valuable insight into the potential cellular mechanisms of CPF toxicity.

Conclusion

The results presented here constitute the first report detailing with the genotoxic effects of CPF in Sertoli cells. CPF induced cytotoxicity in Sertoli cells by changing cell morphologies and functions at applied concentrations. In addition, the reductions in the expression levels of important antioxidant enzymes and the dramatic increase in lipid peroxidation caused by CPF are evidence that it triggers oxidative damage in Sertoli cells. DNA damage calculated by different techniques in Sertoli cells treated with CPF, and the increase in apoptotic cell numbers indicate genotoxic damage. In this context, it is thought that the mechanism underlying the cytotoxicity of CPF in Sertoli cells may be the result of mutagenic and apoptotic effects in cells. CPF causes cytotoxicity in Sertoli cells by changing cell morphologies and functions at applied concentrations.

Footnotes

Author contributions

Yasemin Aydin and Banu Orta-Yilmaz: Project administration, funding acquisition, conceptualization, methodology, investigation, formal analysis, resources, visualization, writing—original draft, and writing—review and editing. Hatice Kurtel and Emine Cakir: Formal analysis, investigation, and methodology.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by Istanbul University Scientific Research Projects (Project No. 36530 and 36577).

ORCID iDs

Yasemin Aydin

Banu Orta Yilmaz

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article.*

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