Mitochondrial impairment related to the immunotoxicity of the herbicides clomazone, glyphosate and sulfentrazone in THP-1 cells

Abstract

Background

Pesticides are indispensable for the cultivation of crops, especially those of economic importance, such as soybeans. Data on the annual use of herbicides in crops show that they correspond to 50%, making it the most used in agriculture.

Aim

Therefore, the aim of this study was to evaluate the toxicity of the three commercial herbicides (clomazone, glyphosate, and sulfentrazone) in THP-1 cells.

Methods

Cells were incubated with 0–5,000 mg/L of the herbicides for 24 h at 37 °C for cytotoxicity evaluation. Additionally, a few toxicological pathways such as reactive species generation, mitochondrial impairment, and interleukin profile, which have been previously involved in the toxicity of pesticides, were also evaluated.

Results

A potential immunotoxic effect of the herbicides on THP-1 cells was observed, especially glyphosate, as it is a powerful agent of cellular immunotoxicity. It was also possible to verify an increase in oxidative stress and IL-8 levels and mitochondrial dysfunction.

Conclusion

All herbicides showed cytotoxic effects in THP-1 monocytes, which were related to mitochondrial impairment.

Graphical Abstract

Graphical Abstract.

Introduction

Pesticides are indispensable for the extensive cultivation of crops, especially those of economic importance, such as soybeans, corn, wheat, etc.¹ Around 2 million tons of pesticides are used annually, among them, herbicides correspond for 50%, followed by insecticides (30%), fungicides (18%), and other types such as rodenticides and nematicides.² Many pesticides are considered harmful to the health of humans and animals or dangerous to the environment. The risk is particularly significant for human health, and occupational and environmental exposure, considering residual amounts, consistently found in food and water. It has been proposed that around 80%–85% of residual pesticides enter the human body via food.³ Prolonged pesticide exposure can result in neurological, reproductive, teratogenic, and immunological disorders.^4–6

The deregulation of the immune system by pesticides has been closely associated with the predisposition to different diseases, especially those associated with immunosuppression or autoimmunity.⁷ There is growing evidence that herbicide exposure affects the immune system by inducing apoptosis in immune cells. Apoptosis occurs to control the immune response and eliminate these compounds from cellular organisms. However, when there is a dysregulation in apoptotic pathways, autoimmune diseases and immunodeficiencies can occur, as shown in recent studies by our research group.^8,9

Therefore, this study aimed to evaluate the toxicity of the herbicides clomazone, glyphosate, and sulfentrazone on the human monocytic cell line THP-1, as well as to contribute to elucidate of the role of toxicological pathways including, reactive species generation, mitochondrial impairment, and interleukin profile, which have been previously involved in the toxicity of different pesticides.

Materials and methods

Chemicals

The herbicides clomazone (Gamit® 360 CS; 99.8% purity), glyphosate (Syngenta®, 620 g/L; 62% purity), and sulfentrazone (Boral® 500 SC; 92.73% purity) obtained commercially were used. Working stock solutions were prepared by diluting the formulations in phosphate-buffered saline (PBS) (pH 7.4) at the following concentrations: clomazone 20,000 mg/L; glyphosate 10,000 mg/L; and sulfentrazone 25,000 mg/L. The stock solutions were filtered through sterile syringe filters and stored at −20 °C.

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin–EDTA, phosphate buffered saline (PBS), sodium chloride (NaCl), 2-mercaptoethanol, amphotericin, penicillin–streptomycin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), ethyl ester perchlorate tetramethylrodamine (TMRE), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), and triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

THP-1 cell culture

The human monocytic leukemia cell line THP-1 has become a common model to estimate the modulation of monocyte and macrophage activities. These cells have been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport.¹⁰ THP-1 cells were cultured following the protocols of American Type Culture Collection (ATCC) and OECD 442E.¹¹ The cells were maintained at 37 °C, under 5% CO₂, and in a humidified atmosphere, in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 U/mL of penicillin, and 100 μg/mL streptomycin. For testing, THP-1 cells were routinely seeded every 2–3 days at a density of 2 × 10⁶ cells/mL and pre-cultured in culture flasks for 24 h, respectively. On the day of testing, cells were harvested from culture flask and resuspended with fresh culture medium at ~2 × 10⁶ cells/mL. Then, cells were distributed into a 96-well flat-bottom plate with 100 μL (2 × 10⁴ cells/well).

MTT reduction assay

The test principle is based on reducing tetrazolium MTT by active reductases, forming formazan crystals only in living cells. The MTT reduction assay was performed as previously described.¹² Concentration-response curves were obtained by incubating the cells with 0–5,000 mg/L clomazone, glyphosate or sulfentrazone for 24 h at 37 °C. Concentrations were selected to cover the whole effect range, from undetectable effects (when compared with negative controls, i.e. 0%) to 100% mortality. Triton X-100 at 1% was used as a positive control, while control cells received only a culture medium. Results were graphically presented as a percentage of cell death vs concentration (mg/L). All concentrations were tested in three independent experiments, with each concentration tested in eight replicates within each experiment.

Challenge for toxicological pathways assays

Subsequent tests were carried out to clarify the mechanisms related to the toxicity of pesticides in this cell model, testing three different concentrations for each test substance. Such concentrations were: 300, 500, and 1,000 mg/L (around 1,2, 2.0 and 4.2 mM) for clomazone; 50, 100, and 150 mg/L (around 0.3, 0.6 and 0.8 mM) for glyphosate; and 100, 300 and 500 mg/L (around 0.2, 0.7 and 1.3 mM) for sulfentrazone, which correspond approximately to the EC₂₅, EC₅₀, and EC₇₅, according to MTT concentration-response curves.

Measurement of intracellular reactive oxygen (ROS) and nitrogen (RNS) species

The intracellular reactive oxygen (ROS) and nitrogen (RNS) species production was monitored with the DCFH-DA assay, as previously described.¹² After 24 h of seeding, cells were incubated with 10 μM DCFH-DA for 30 min at 37 °C, protected from light. DCFH-DA was initially prepared as a 4 mM stock solution in DMSO and made up to the final concentration in fresh culture medium (ensuring that the final concentration of DMSO did not exceed 0.05%) immediately before each experiment. The cells were then rinsed with PBS and incubated with the herbicides at 37 °C for 24 h under a humidified atmosphere of 5% CO₂. Tert-butyl hydroperoxide (TBHP) at 500 μM was used as positive control. After 24 h incubation, fluorescence was recorded in a microplate reader (FLUOstar® OPTIMA Offenburg, Germany) set to 485 nm excitation and 530 nm emission. The data obtained were normalized to negative control on a plate-by-plate basis and calculated as a fold increase over the control conditions of three independent experiments, with each concentration tested in eight replicates within each experiment.

Assessment of mitochondrial membrane potential (Δψm)

The assessment of mitochondrial integrity was performed by measuring the inclusion of TMRE.¹³ After 24 h of seeding, cells were incubated with three concentrations of each herbicide at 37 °C for 24 h, under a humidified atmosphere of 5% CO₂. At the end of the incubation period, the medium was replaced with PBS containing 2 mM TMRE and incubated at 37 °C for 30 min, protected from light. As TMRE is a non-water-soluble powder, a 2 μM stock solution was prepared in DMSO and stored protected from light. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 5 μM, a decoupler of oxidative phosphorylation, was used as positive control. Afterwards, the medium was aspirated and replaced by PBS. The fluorescence was recorded in a microplate reader (FLUOstar® OPTIMA Offenburg, Germany) set to 544 nm excitation and 590 nm emission. The data obtained were normalized plate by plate to the values of the respective controls and calculated as the percentage of control conditions from three independent experiments, with each concentration tested in eight replicates within each experiment.

Interleukin profile assessment

The evaluation of the interleukin profile was determined using the Human Inflammatory Cytokine Kit BD™ CBA (cytometric beads array) for the quantification of interleukin-8 (IL-8), interleukin-1β (IL-1β), interleukin-6 (IL −6), interleukin-10 (IL-10), tumor necrosis factor (TNF) and interleukin-12p70 (IL-12p70). Cells were seeded in 96-well plates at a density of 2×10⁴ cells/mL. The cells were incubated with three concentrations of each herbicide at 37 °C for 24 h, under a humidified atmosphere of 5% CO₂. Assessments of interleukin profiles were read on the FACSCanto II (BD) flow cytometer. The results were generated in graphs and tables using the FCAP Array v3 (BD) software. Kit performance was optimized for analysis of proteins in lysed supernatants from cell culture with the Bicinchoninic Acid Protein Assay Kit (Sigma) and adjusted to 562 nm.

Statistical analysis

GraphPad Prism 5 was used for statistical analyses. The statistical comparisons between groups were performed using one-way ANOVA, followed by Bonferroni’s post-hoc test. The normality of data distribution was assessed using the Kolmogorov–Smirnov test. Data are expressed as mean ± standard error of the mean (SEM) at least three independent experiments, with P ≤ 0.05 considered statistically significant.

Results

Cytotoxicity

The herbicides clomazone, glyphosate, and sulfentrazone produced concentration-dependent cytotoxic effects (Fig. 1). Significant differences in the IC₅₀ values of glyphosate (IC₅₀ 71.71 mg/L or 0.42 mM) compared to sulfentrazone (IC₅₀ 242.8 mg/L or 0.62 mM) and clomazone (IC₅₀ 576.0 mg/L or 2.4 mM) were found, where glyphosate was the most cytotoxic herbicide tested. No significant differences were observed for clomazone and sulfentrazone IC₅₀. A summary of the IC₅₀ values is provided in Table 1.

Fig. 1.

Concentration-response curves of the tested pesticides after 24 h incubation in THP-1 cells at 37 °C. Cell mortality was evaluated by the MTT reduction assay. Data are presented as percentage of cell death relative to the respective negative controls, after normalization to the positive control. Curves were fitted using least squares as the fitting method. Three independent experiments were performed (eight replicates tested for each concentration within each experiment).

Table 1.

EC₅₀ values data were obtained by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 24 h of incubation in THP-1 cells.

Herbicide	EC50 (mg/L)
Clomazone	576.0
Glyphosate	71.71
Sulfentrazone	242.8

The cytotoxicity curves were fitted using least squares as the fitting method.

Production of ROS and RNS

In this study, the production of ROS and RNS in THP-1 was significantly increased by incubation with 500 μM TBHP (P < 0.001, ANOVA/Bonferroni, Fig. 2), used as a positive control, validating the assay. Only 500 mg/L sulfentrazone significantly (P < 0.0001, ANOVA/Bonferroni) increased the reactive species formation.

Fig. 2.

Reactive species of oxygen and nitrogen (ROS/RNS) production, measured by the DCFH-DA assay, in THP-1 cells after 24 h incubations at 37 °C. Results are expressed as percentage control ± SEM. Three independent experiments were performed (eight replicates for each concentration within each experiment). Statistical comparisons were made using one-way ANOVA/Bonferroni post hoc test (^***P < 0.001 vs. control). Clo (clomazone); Gly (glyphosate); Sul (sulfentrazone); TBHP (tert-butyl hydroperoxide; positive control at 500 μM).

Mitochondrial membrane potential

As shown in Fig. 3, a significant decrease in Δψm was observed for the positive control CCP and at all clomazone concentrations and 100 and 150 mg/L glyphosate (P < 0.001, ANOVA/Bonferroni). Interestingly, at all tested concentrations, sulfentrazone increased in Δψm (P < 0.01 ANOVA/Bonferroni), indicative of mitochondrial hyperpolarization.

Fig. 3.

Mitochondrial membrane potential (Δψm) production, measured by the TMRE assay, in THP-1 cells after 24 h incubations at 37 °C. Results are expressed as percentage control ± SEM. Three independent experiments were performed (eight replicates for each concentration within each experiment). Statistical comparisons were made using one-way ANOVA/Bonferroni post hoc test (^**P < 0.01; ^***P < 0.001 vs. control). Clo (clomazone); Gly (glyphosate); Sul (sulfentrazone); CCCP (carbonyl cyanide chlorophenylhydrazone; positive control at 5 μM).

Interleukins profile

Levels of the pro-inflammatory cytokine IL-8 were significantly increased, compared to control, in the supernatant of THP-1 cells incubated with glyphosate (Fig. 4). This result made clear the effect of the herbicide glyphosate as a powerful agent of cellular immunotoxicity. The levels were below the detection limit for the other interleukins (IL-1β, IL-6, IL-10, TNF, and IL-12p70).

Fig. 4.

The levels of interleukin-8 (IL-8), in the supernatant of THP-1 cells after 24 h incubations at 37 °C. Results are expressed as percentage control ± SEM. Three independent experiments were performed (eight replicates for each concentration within each experiment). Statistical comparisons were made using one-way ANOVA/Bonferroni post hoc test (^***P < 0.001 vs. control). Clo (clomazone); Gly (glyphosate); Sul (sulfentrazone).

Discussion

THP-1 cells are derived from the peripheral blood of a 1-year-old male patient with acute monocytic leukemia.¹⁴ This cell line, in both; monocyte and macrophage states, has been widely used to study chemically induced immune responses induced by chemicals.^15,16 Monocytes and macrophages belong to the innate immune compartment. The main roles are recognizing foreign pathogens, producing pro-inflammatory chemokines and cytokines, and recruiting effector cells to the site of infection and anti-inflammatory cytokines when the infection is under control.¹⁷

Our results confirm previous evidence of glyphosate cytotoxicity in human Raji hematologic cell lines.¹⁸ Concentration-dependent cytotoxic effects were also observed for glyphosate and the commercial formulation Roundup® Bioflow (360 g/L) in L929 and Caco2 cells.¹⁹ In March 2015, the WHO decided to change the classification of glyphosate to Category 2A, as probably carcinogenic to humans, due to the reassessment of the safety profile of this herbicide.²⁰ In addition to frequent and excessive use of glyphosate in agriculture, use in residential and urban areas such as sidewalks and parking lots is frequent.²¹ Notably, glyphosate is the most applied pesticide globally, and exposure and sales to this substance continue to increase.²² Therefore, this study used glyphosate as a positive control for the other pesticides.

For the herbicide sulfentrazone, a study with Allium cepa seeds verified cytotoxic effects by inducing nuclear fragmentation and inhibition of cell division at 6 g/L of the herbicide.²³ On the other hand, a study by Santi et al.²⁴ reported insufficient data on the cytotoxic effects of clomazone. Nevertheless, in vitro studies are required to better understand clomazone and sulfentrazone toxic mechanisms in humans.

Oxidative stress causes an imbalance between the pro-oxidant and antioxidant systems, causing cell damage. When increased production of these species overcomes antioxidant defenses, cellular homeostasis is affected. In this study, the results found for sulfentrazone were similar to those found by Li et al.,²⁵ who observed an increase in the production of reactive species in the in vivo model Eisinia fetida after using sulfentrazone. This indicates that oxidative stress is an important pathway for sulfentrazone toxicity. The results found for glyphosate follow previous studies from our group, which also do not demonstrate alteration in the production of reactive species in HepG2 cells after 48 h of incubation.¹² In contrast to our results, it is reported that the herbicide clomazone increased the rate of oxidation of DCFH DA in the whole blood of common carp exposed for seven days at 5 mg/L^-1.26.

Mitochondria participate in several vital cell functions, mainly energy production through ATP generation and Ca²⁺ buffering. Previously, we demonstrated that the herbicide glyphosate depolarized mitochondria in HepG2 cells.¹² As observed by our results, the mitochondrial function also was severely affected by 125 μM clomazone, which significantly reduced ATP levels in mitochondria isolated from the chest of bees (Apis mellifera).²⁷ Stable ΔΨm is required for mitochondrial function, while hyperpolarization or depolarization through the excessive movement of ions (i.e. Ca²⁺, H⁺, and ROS) across mitochondrial membranes indicates a pathophysiological state. Cell depolarization or mitochondrial hyperpolarization results in apoptotic and/or necrotic stimuli.^28,29 Mitochondrial hyperpolarization was also verified in a study by Jiang et al.³⁰ with zebrafish embryos exposed to 0.01, 0.1, and 0.4 mg/L⁻¹ of sulfentrazone for 5 and 30 days. In addition, there was an increase in mitochondrial complex IV and cytochrome c oxidase activities after exposure at both times. This is the first exposure scenario where the herbicide clomazone causes mitochondrial depolarization, and the herbicide sulfentrazone causes mitochondrial hyperpolarization after 24 h incubation of THP-1 cells.

Interleukin levels in the cell supernatant were measured to estimate immune responses. Alternatively known as CXCL8, IL-8 is a pro-inflammatory CXC chemokine secreted by several cell types, including monocytes.³¹ It is released in response to an inflammatory stimulus. Therefore, it plays an essential role in inflammation and wound healing and can recruit T cells and nonspecific inflammatory cells at sites of inflammation.³² As observed in our study, increased IL-8 expression was also observed in the gills of fish treated with commercial glyphosate at 52.08 and 104.15 mg/L⁻¹ for seven days, indicating that this herbicide induces an inflammatory response.³³ Interestingly, this is the first study to report an interleukins profile after incubating cells with clomazone and sulfentrazone.

Conclusion

The results revealed a cytotoxic effect of the herbicides clomazone, glyphosate, and sulfentrazone on THP-1 immune cells after 24 h incubation. All herbicides evaluated showed cytotoxic effects under our experimental conditions, but glyphosate, one of the most applied herbicides in the world, was identified as the most potent. Among the mechanisms related to the cytotoxicity of the herbicides in immune cells, mitochondrial impairment is an essential target and should be investigated in depth.

Author contributions

Larissa Vivan Cestonaro: investigation, formal analysis and writing—original draft preparation.

Artur Christian Garcia da Silva: investigation, formal analysis and writing—review & editing.

Solange Cristina Garcia: resources, funding acquisition and writing—review & editing.

Marize Campos Valadares: conceptualization, resources, funding acquisition and writing—review & editing.

Marcelo Dutra Arbo: conceptualization, project administration, resources, funding acquisition and writing—review & editing.

Funding

This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES Foundation, financial code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação de Amparo a Pesquisa do Rio Grande do Sul (FAPERGS, 19/2551-0001900-9).

 

Conflict of interest statement: The authors declare that there is no conflict of interest.