Flumetralin based plant growth regulator used in tobacco cultivation induces immune and cytotoxicity in vitro

Abstract

Tobacco production lasts about 10 months and various pesticides are used, including growth inhibitors, which flumetralin is the most used. This is an herbicide that acts as a synthetic and plant growth regulator. Therefore, this work aimed to evaluate the toxicity of the growth regulator flumetralin in RAW 264.7 and 3T3 cell lines. Cytotoxicity was assessed by MTT reduction and neutral red uptake assays after 24 h of incubation with flumetralin. Mitochondrial integrity, production of reactive species and cytokine profile were evaluated in both cell lines. Furthermore, NO production was evaluated in RAW 264.7 cells, while comet assay was evaluated in 3T3 cells. An increase in reactive species production was observed in both cell lines. In RAW 264.7 cells were observed an increase in mitochondrial membrane potential, while 3T3 cells presented a mitochondrial depolarization. At all tested concentrations, flumetralin increased TNF-α levels and decreased IL-10 levels in RAW 264.7, and increased TNF and IL-1ß in 3T3 cells. In addition, at all tested concentrations, flumetralin induced DNA damage in 3T3 cells. It was possible to observe the cytotoxic effect of flumetralin on the tested cell lines, as well as a possible generation of an inflammatory response and immune deregulation.

Introduction

Tobacco farming is an agricultural crop that has a high production chain, being cultivated in more than 130 countries. Since 2020, Brazil occupies third place in the export ranking of this crop. Brazilian tobacco is known for high quality, guaranteed supply and for being more competitive in the international market due to the fact that it has cheaper labor when compared to other countries. In the southern region of Brazil, tobacco is cultivated in more than 500 municipalities, involving around 140 thousand small producers.^1–3

​ The tobacco production process is long, taking around 10 mo, from planting to harvesting, and the production of this crop requires exhaustive working hours from producers, in addition to the need to use various pesticides and anti-sprouting agents. An important factor in tobacco cultivation is the need to carry out “disbudding” to prevent nutritive substances from being dispersed between the different organs of the plant through the leaf and nutrient competition occurring between the lateral shoots and the tobacco leaves. These substances, when accumulated in the leaves of the plant, help in the growth of stronger plant, therefore, the elimination of buds is necessary for the quality of the leaves.^4–6

The disbudding process occurs 2 to 4 times throughout the planting cycle. Therefore, to reduce costs and hours worked, producers started using growth inhibitors, also known as anti-sprouting agents. However, their use can cause several risks to the health of the farmer and people who live close to the crops, such as respiratory diseases, cancer, changes in the immune and reproductive system, endocrine changes, development of diabetes, in addition to various other damages to the body.^7,8 In tobacco farming, the most used anti-budding agent is Primeplus®, which has flumetralin as its active ingredient (Fig. 1A). Flumetralin is a synthetic plant growth regulator, belonging to the 2,6-dinitroaniline class, and is widely used in tobacco farming to control the growth of axillary buds in the tobacco plant.⁹

Fig. 1.

Flumetralin structural formula A), adapted from sigma Aldrich, 2023) and flumetralin concentration-response curves tested after 24 h of incubation at 37 °C in RAW 264.7 B) and 3T3 C) cells. The black curve depicts cell mortality assessed by the MTT reduction assay and the red curve depicts mortality using the neutral red (NR) uptake assay.

Flumetralin poisoning can occur both directly through dermal contact in the workplace and during the application of the product, or indirectly, for example, when this substance comes into contact with the soil and drinking water, in addition to people who smoke. In 2014, flumetralin was classified by Brazilian National Health Surveillance Agency (ANVISA) as not likely to be carcinogenic and also as not likely to be genotoxic to humans as long as the instructions on the package insert were followed, however, in the same year it entered the Pesticides Action Network - International List of Highly Hazardous Pesticides.^10–13 This list is not based solely on the acute toxicity and it also does not consider only carcinogenicity or lethality such as ANVISA, it considers factors such as bioaccumulation, environmental persistence, toxicity to aquatic organisms, chronic effects, etc and has a very broad focus, considering risks to human health and the environment under real conditions of use, including the effects of the formulated product, and not just the pure active substance. Therefore, tis work aimed to evaluate the toxicity of a commercial flumetralin formulation in RAW 264.7 and 3T3 cells.

Materials and methods

Chemicals

The commercial product Primeplus®, which has 12.5% w/v of the active ingredient flumetralin, in addition to 25% w/v cyclohexanone, 28% w/v solvent naphta (petroleum), and 87.5% w/v other ingredients that are not listed was used. DMEM (Dulbecco’s Modified Eagle) medium, fetal bovine serum (FBS), amphotericin, penicillin, streptomycin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), methylrhodamine perchlorate tetraethyl ester (TMRE), 3-amino-7-dimethylamino-2-methylphenazine hydrochloride (neutral red - NR), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), sulfanilamide, N-(1-naphthyl)ethylenediamine dihydrochloride (NED), triton X-100, and trypan blue were obtained from Sigma-Aldrich (St. Louis, MO, USA).

RAW 264.7 cell culture

The RAW 264.7 cell line is originated from mouse macrophages.¹⁴ The cells were cultivated in sterile culture 75 cm² flasks with DMEM, supplemented with 20% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 250 μg/mL amphotericin and were maintained in a humidified atmosphere with 5% CO₂ at 37 °C. The culture medium was changed every 2–3 days and when the cells reached 70–80% confluence, they were subcultured.

3T3 cell culture

The 3T3 cell line originates from fibroblasts and is widely used for toxicological evaluations.¹⁵ The cells were maintained in sterile culture 75 cm² flasks with DMEM culture medium with a high concentration of glucose, supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 250 μg/mL amphotericin and were maintained in a humidified atmosphere with 5% CO₂ at 37 °C. The culture medium was changed every 2–3 days and when the cells reached 70–80% confluence, they were subcultured.

Cytotoxicity assay

Cytotoxicity was evaluated using MTT reduction and Neutral Red uptake assays.

MTT reduction assay

The MTT reduction assay was performed as described by Cestonaro et al. (2023).¹⁶ This assay measures the activity of cellular dehydrogenases, an indicator of metabolically active cells. In the RAW 264.7 cell line, 40,000 cells/mL were seeded in 96-well plates, while in the 3T3 cell line, 10,000 cells/mL were seeded in 96-well plates. Stock concentration of 2.5 mg/L flumetralin was prepared in PBS and stored at 20 °C. Working solutions were prepared by diluting the stock solution in cell culture medium. Cells were incubated with 0–1.0 mg/L flumetralin for 24 h at 37 °C to obtain concentration-response curves in both cell lines. As a positive control, 1% triton x-100 was used. After incubation with flumetralin for 24 h, the medium was removed and replaced with PBS containing 0.5 mg/mL MTT and the cells were incubated at 37 °C for 2 h. DMSO was used to dissolve the formed formazan and absorbance was measured at 550 nm in a multiwell plate reader (SpectraMax® MiniMax™ 300, CA, USA). Results were presented graphically as percentage of cell death versus concentration. All concentrations were tested in three independent experiments with each concentration tested in four replicates within each experiment.

Neutral red uptake assay

The NR uptake test was performed as described by Cestonaro et al. (2023).¹⁶ In the RAW 264.7 cell line, 40,000 cells/mL were seeded in 96-well plates, while in the 3T3 cell line, 10,000 cells/mL were seeded in 96-well plates. Stock concentration of 2.5 mg/L flumetralin was prepared in PBS and stored at 20 °C. Working solutions were prepared by diluting the stock solution in cell culture medium. Cells were incubated with 0–1.0 mg/L flumetralin for 24 h at 37 °C to obtain concentration-response curves in both cell lines. As a positive control, 1% triton x-100 was used. At the end of 24 h of incubation, both cell cultures had the medium replaced with fresh medium containing 50 μg/mL of NR and the cells were incubated at 37 °C for 3 h. Subsequently, cells were lysed with a mixture of 50% ethanol/1% glacial acetic acid solution and absorbance was measured at 540 nm in a multiwell plate reader (SpectraMax® Mini Max™ 300, CA, USA). The percentage of cell death relative to the control was used as a measure of cytotoxicity. All concentrations were tested in three independent experiments with each concentration tested in four replicates within each experiment.

Challenge for other toxicological assays

Subsequent tests were carried out to understand some mechanisms related to flumetralin toxicity, testing three different concentrations corresponding to three different levels of cytotoxicity, EC20, EC40 and EC60 according to the MTT concentration-response curve. The concentrations tested here were, respectively, 0.085, 0.195 and 0.286 mg/L of flumetralin for RAW 264.7 cells and 0.055, 0.068 and 0.079 mg/L for 3T3 cells. These are environmentally relevant concentrations, a study carried out by Fan et al (2018)¹⁷ detected the presence of flumetralin in soil at concentrations such as 0.43 to 3.9 mg/kg. This study also points that flumetralin is persistent in the environment and is not considered rapidly degradable.

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

Intracellular production of reactive oxygen and nitrogen species was assessed using the dichloro-dihydrofluorescein diacetate assay, as previously described by Conte et al (2022).¹⁸ Cells were seeded as described for cytotoxicity, in 96-well plates, with a final volume of 100 μL of culture medium. After 24 h, the cells were incubated with 10 μM DCFH-DA for 30 min at 37 °C protected from light. A stock solution of 4 mM DCFH-DA was prepared in DMSO and brought to final concentration in fresh culture medium before each experiment. After incubation, cells were rinsed with PBS and incubated with flumetralin at EC20, EC40 and EC60 at 37 °C. TBHP (tert-butyl hydroperoxide) 500 μM was used as a positive control. After 24 h of incubation, fluorescence was recorded on a microplate reader (SpectraMax® Mini Max™ 300, CA, USA) set to 485 nm excitation and 530 nm emission. Three independent experiments were performed, with each concentration tested in four replicates within each experiment.

Assessment of mitochondrial membrane potential (Δψm)

TMRE (tetramethyl rhodamine ethyl ester) is a positively charged red-orange dye that permeates cells and accumulates in active mitochondria (which in turn are negatively charged). RAW 264.7 cells were seeded in 96-well plates at 40,000 cells/mL, while the 3T3 cell line was seeded at 10,000 cells/mL in 96-well plates. After 24 h, the cells were incubated with their respective EC20, EC40 and EC60 of flumetralin at 37 °C for 24 h. At the end of incubation, the medium was replaced with PBS containing 2 mM TMRE and incubated for 30 min at 37 °C, protected from light. A stock solution of 2 mM TMRE was prepared in DMSO and stored protected from light at −20 °C. The positive control used was CCCP (carbonyl cyanide m-chlorophenyl hydrazone) 100 mM. Fluorescence was measured at 37 °C on a microplate reader (SpectraMax® MiniMax™ 300, CA, USA) set to 544 nm excitation and 590 nm emission.¹⁸ The data obtained were calculated as the percentage of control conditions from three independent experiments with each concentration tested in four replicates within each experiment.

Griess test

As described by Cestonaro et al. (2023),¹⁶ the Griess test is used to measure the conversion of NO to nitrite. RAW 264.7 cells were seeded in 96-well plates at a density of 40,000 cells/mL. After 24 h, cells were incubated with EC20, EC40 and EC60 of flumetralin at 37 °C. Then, 5,000 ng/mL of LPS was added to the plates and incubated for the last 24 h at 37 °C. Nitrite levels were measured in 100 μL of supernatant, which were transferred to another plate and mixed with 100 μL of Griess reagent (2% sulfanilamide and 0.2% NED 1:1). After 20 min of incubation at 37 °C, absorbance was measured at 540 nm on a microplate reader (SpectraMax® MiniMax™ 300, CA, USA). Sodium nitrite was used as standard in the calibration curve. Three independent experiments, with each concentration tested in three replicates, were performed.

Cytokine profile

RAW 264.7 cells were seeded in 96-well plates at a density of 40,000 cells/mL. After 24 h, cells were incubated with EC20, EC40 and EC60 of flumetralin at 37 °C. The culture medium was used to measure the concentrations of tumor necrosis factor (TNF) and interleukin-10 (IL-10) through the sandwich ELISA test, according to the manufacturer’s instructions (R&D Systems - DuoSet®). In the same way, 3T3 cells were seeded in 24-well plates at a density of 400,000 cells/mL and incubated with EC20, EC40 and EC60 of flumetralin at 37 °C. The culture medium was used to measure the concentrations of tumor necrosis factor (TNF) and interleukin-1β (IL-1β) through the sandwich ELISA test, according to the manufacturer’s instructions (R&D Systems - DuoSet®).

Comet assay

The comet assay was performed with the 3T3 cell line using 24-well plates. The cells were seeded at a density of 400,000 cells/mL with a final volume of 0.5 mL of medium and, after 24 h, they were incubated with flumetralin at concentrations equivalent to EC20, EC40 and EC60. Doxorubicin 50 μM was used as positive control. After 24 h of incubation at 37 °C, the cells were harvested with 0.05% trypsin/EDTA and centrifuged (400 g, 10 min). The cell pellets obtained were resuspended in low-melting-point agarose (0.75%, 90 μL) and two aliquots of 50 μL were placed on slides previously coated with 1% normal-melting-point agarose. The slides containing the samples were placed in tanks with lysis solution (2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Tris–HCl, 10% DMSO, pH 10; 1% Triton X100) at 4 °C for 24 h in the dark. Subsequently, the slides were incubated with alkaline electrophoresis running buffer (1 mM Na₂EDTA, 300 mM NaOH, pH 13.0) for 20 min at 4 °C and then electrophoresis was performed for 20 min at 25 V and 300 mA. Neutralization of the slides with 0.4 M Tris–HCl was performed in the dark for 15 min. DNA fixation was done in absolute alcohol for 5 min in the dark, and the slides were dried overnight.¹⁸ GelRed® (20 μg/mL) was used to stain the slides and DNA migration was observed in at least 100 cells using 400x magnification on a fluorescence microscope (Leica®, Germany) with a 510–550 nm excitation filter, connected to a camera.

In parallel to the comet assay, an identical slide was prepared, lysed, fixed and stained, but not subjected to electrophoresis, to perform the low molecular weight DNA diffusion assay (LMW-DNA). This test assesses cytotoxicity and detects pre-lethal DNA breaks. In addition, it helps to verify whether comet responses are affected only by cytotoxicity, avoiding false negative/positive results due to the formation of cytotoxic compounds.¹⁹ Three independent experiments were performed and the images were evaluated using the Comet ScoreTM program, obtained from the public domain (http://www.tritekcorp.com/products_cometscore.php). DNA damage was quantified by measuring the percentage of DNA in the comet tail (% DNA).

Statistical analysis

Statistical analyzes were performed in GraphPad Prism 5. Statistical comparisons were performed using one-way ANOVA followed by Bonferroni posthoc test. Data were expressed as mean ± standard error of the mean with significance accepted at P < 0.05.

In silico ADMETox prediction

Prediction of pharmacokinetic (absorption, distribution, metabolism, excretion) and pharmacodynamic (toxicity endpoints) properties of flumetralin were performed with SwisADME,²⁰ SuperPred 3.0,²¹ Pred-hERG,²² Pred-Skin,²³ eMolTOX,²⁴ and ProTox3.²⁵

Results

Flumetralin induced a concentration-dependent cytotoxicity in RAW 264.7 and 3T3 cells

A concentration-response analysis was carried out by incubating the RAW 264.7 cells with 0–1.0 mg/L flumetralin for 24 h in both cell lines. Figure 1B demonstrates the concentration-response curves obtained through the MTT reduction and the NR uptake assays for RAW 264.7 cells. It was observed a concentration-dependent cell death and the calculated EC50 values obtained by MTT reduction and NR uptake assays were, respectively, 0.237 and 0.253 mg/L in Raw 264.7 cells (table 1). Figure 1C presents the concentration-response curves obtained through the MTT reduction and the NR uptake assays for 3T3 cells. The obtained EC50 values were, respectively, 0.075 and 0.068 mg/L (table 1).

Table 1.

EC50 values of flumetralin after 24 h of incubation in RAW 264.7 and 3T3 cells.

Cell line	EC50 MTT (mg/L)	EC50 NR (mg/L)
RAW 264.7	0.237 mg/L	0.253 mg/L
3T3	0.075 mg/L	0.068 mg/L

Flumetralin induced free radicals production

ROS/RNS production was measured using the DCFH-DA assay in RAW 264.7 and 3T3 cell lines after 24 h of incubation. A significant increase (P < 0.01, ANOVA/Bonferroni) of reactive species was observed in RAW 264.7 cells at concentrations corresponding to EC40 and EC60, compared to control (Fig. 2A). In 3T3 cells, all concentrations showed a significant increase (P < 0.001, ANOVA/Bonferroni) in the production of reactive species (Fig. 2B). The positive control (TBHP) also showed a significant increase in the production of reactive species (P < 0.05, ANOVA/Bonferroni).

Fig. 2.

Reactive species of oxygen and nitrogen (ROS/RNS) production, measured by the DCFH-DA assay, in RAW 264.7 A) and 3T3 B) cells after 24 h incubation with flumetralin and C) NO levels in RAW 264.7 cells after exposure to flumetralin for 24 h. the values are expressed as a percentage of the control ± standard error of the mean (n = 3 independent experiments carried out in eight replicates). Statistical comparisons were made by ANOVA/Bonferroni (^*P < 0.05; ^**P < 0.01; ^***P < 0.001 vs control).

The growth inhibitor flumetralin affects mitochondrial function

Mitochondria are important for several vital functions of the cell, mainly to the energy production, through the generation of ATP. Therefore, to investigate whether flumetralin alters mitochondrial function, Δψm was evaluated. Significant mitochondrial depolarization (P < 0.05, ANOVA/Bonferroni) was observed in cells incubated with CCCP (positive control). In RAW 264.7 cells, a significant (P < 0.001, ANOVA/Bonferroni) increase in Δψm was observed at EC60, characterizing hyperpolarization (Fig. 3A). Interestingly, in the 3T3 cell line (Fig. 3B), it was noted a significant decrease, i.e. depolarization, in Δψm at all tested flumetralin concentrations (P < 0.001, ANOVA/Bonferroni).

Fig. 3.

Mitochondrial membrane potential (Δψm) measured by the TMRE assay, in RAW 264.7 A) and 3T3 B) cells after 24 h incubation with flumetralin. The values are expressed as a percentage of the control ± standard error of the mean (n = 3 independent experiments carried out in eight replicates). Statistical comparisons were made by ANOVA/Bonferroni (^*P < 0.05; ^***P < 0.001 vs control).

Flumetralin alters inflammatory response in RAW 264.7 cells

The NO test was used to evaluate the effect of flumetralin on the response of RAW 264.7 cells to an inflammatory stimulus. No significant difference was found, under our experimental conditions, in NO levels when compared to the control after 24 h of incubation (Fig. 2C).

The levels of pro- and anti-inflammatory cytokines were evaluated in the RAW 264.7 cell line to estimate the cells’ immune responses. As shown in Fig. 4, there was a significant increase (P < 0.05, ANOVA/Bonferroni) in the levels of the pro-inflammatory cytokine TNF at all tested concentrations tested. On the other hand, there was a significant decrease (P < 0.01, ANOVA/Bonferroni) in the anti-inflammatory IL-10 levels.

Fig. 4.

Levels of tumor necrosis factor (TNF, A) and interleukine 10 (IL-10, B) in RAW 264.7 cells after incubation with flumetralin for 24 h. the values are expressed as mean ± standard error of the mean (n = 3 independent experiments carried out in eight replicates). Statistical comparisons were made by ANOVA/Bonferroni (^*P < 0.05; ^**P < 0.01; ^***P < 0.001 vs control).

The levels of cytokines were also evaluated in the 3T3 cell line. As shown in Fig. 5, there was a significant increase (P < 0.05, ANOVA/Bonferroni) in TNF levels at a concentration equivalent to EC40. Likewise, IL-1β levels also demonstrated a significant increase at the same concentration.

Fig. 5.

Levels of tumor necrosis factor (TNF, A) and interleukine 1beta (IL-1ß, B) in 3T3 cells after incubation with flumetralin for 24 h. the values are expressed as mean ± standard error of the mean (n = 3 independent experiments carried out in eight replicates). Statistical comparisons were made by ANOVA/Bonferroni (^*P < 0.05; ^**P < 0.01 vs control).

Flumetralin induced DNA damage in 3T3 cells

The comet assay is considered a sensitive method to detect strand breaks and alkaline-labile sites in the nucleus of eukaryotic cells. In this context, it is used to evaluate DNA damage induced by genotoxic agents, being used as a biomarker for genotoxicity. In our study, it was possible to observe through the comet assay that flumetralin significantly increases the percentage of DNA in the tail, both in the alkaline assay (Fig. 6A) and in the LMW-DNA assay (Fig. 6B). Representative images of comet assay and LMW DNA are shown in supplementary figs S1 and S2, respectively.

Fig. 6.

Comet A) and low molecular weight (LMW) DNA diffusion B) assays in 3T3 cells after 24 h incubation with flumetralin. The values are expressed as mean ± standard error of the mean (n = 3 independent experiments carried out in duplicates). Statistical comparisons were made by ANOVA/Bonferroni (^*P < 0.05; ^**P < 0.01; ^***P < 0.001 vs control).

In silico prediction points to genotoxicity and immunotoxicity

Different ADMETox aspects of flumetralin were inspected by employing varied computational prediction tools. Full reports are shown in Supplementary File 1. Deserving special attention is the high confidence prediction of skin sensitization in multiple models, including inflammation induction. In addition to that, tissue damage, genotoxicty, endocrine disruption, and immunotoxicity were all predicted with high confidence.

Discussion

Flumetralin toxicological studies are very limited, however studies carried out with other herbicides from the dinitroaniline class report several toxic effects, such as endocrine deregulation,²⁶ oxidative damage,²⁷ genotoxicity,^28,29 and mutagenicity.³⁰ However, Giglio and Vommaro (2022)³¹ emphasize that studies for this class of pesticides are still limited and need to be expanded to commercial formulations. In this study, we evaluated the toxicity of flumetralin, which belongs to the dinitroaniline class in the commercial formulation Primeplus®, in RAW 264.7 and 3T3 cells.

Cytotoxicity has been reported for the dinitroaniline herbicide pendimethalin in ovarian cells (CHO)³² and FRTL-5 cell line, derived from rat thyroid.³³ Here, we described, for the first time, the cytotoxicity of flumetralin in murine macrophages RAW 264.7 cells and in healthy 3T3 mouse embryonic fibroblasts. In spite of being different in vitro models, the obtained EC50 values were very close, with the healthy 3T3 cells being a quite more sensitive in vitro model.

As already reported, oxidative damage seems to be a toxicological pathway of the dinitroanilines. Reactive oxygen and nitrogen species are directly related to cellular oxidative stress and are constantly produced, under normal conditions, however, on a small scale. When this production occurs in excess, as observed in both RAW 264.7 and 3T3 cells, it can cause various damages to the integrity of macromolecules such as DNA, proteins and cell membrane lipids.^34,35

The main sources of ROS and RNS in cells are environmental toxins and the oxidative phosphorylation process and emphasize that mitochondrial dysfunction and stress are directly related in the pathophysiology of many diseases.³⁶ Therefore, the joint assessment of free radical production and ΔΨm allows obtaining important information about the physiological state of the cell, as well as the function of mitochondria. In our study, we observed that the cell lines tested showed different responses for mitochondrial activity, when incubated with flumetralin. RAW 264.7 cells showed mitochondrial hyperpolarization, while 3T3 cells showed mitochondrial depolarization. When mitochondrial membrane potential depolarization occurs, it consequently increases the production of free radicals and causes the release of pro-apoptotic factors, as rupture of the outer membrane can occur, causing leakage of proteins from the intermembrane space, including cytochrome C, procaspase and other factors that trigger cell apoptosis or cause necrosis.³⁷ When hyperpolarization occurs, as observed in RAW 264.7 cells, ATP is produced in greater quantities to keep cells alive, however, in the initial process of apoptosis, hyperpolarization can also occur to increase ATP production in order to stimulate the apoptotic pathways and subsequently depolarize to begin the process of apoptosis, with the breakdown of mitochondrial membrane potential.^38,39 Interestingly, this effect on mitochondria could be related to the cytotoxicity, where 3T3 cells, the most sensitive in vitro model, seem more affected by flumetralin, in an apoptotic or necrotic cell death process. On the other side, RAW 264.7 cells were more resistant, with higher EC50 values, and a flumetralin induced hyperpolarization, which represents the first step to increase ATP synthesis to stimulate apoptotic pathways.

Studies have shown that different pesticides can induce immune dysfunctions. Immunosuppression is described as the main mechanism in which pesticides exert immunomodulatory activity, which can therefore increase susceptibility to diseases as it affects immunocompetence.^40–42 In the immune system, nitric oxide (NO) performs several immunological functions, such as activating macrophages, allowing them to maintain or control various pathogens dependent on nitric oxide synthase 2 (NOS2). This enzyme also regulates T cells, B cells and myeloid-derived suppressor cells.^43,44 However, no alterations of NO levels were observed. Instead, changes in cytokine profile were observed after incubation of RAW 264.7 and 3T3 cells with flumatralin.

TNF is one of the main pro-inflammatory cytokines in the early phase immune response, in addition, it can modulate the inflammatory response and is important in the repair process.⁴⁵ Macrophages can also secrete TNF as a result of cellular oxidative stress.⁴⁶ Therefore, as ROS/RNS levels increased in both cell lines, an increase in TNF levels was also expected.

On the other hand, IL-10 is an anti-inflammatory cytokine, crucial in preventing autoimmune diseases and inflammatory pathologies. IL-10 works at different stages of immune response in order to exert its role and inhibit inflammatory pathologies.⁴⁷ The decreased levels of IL-10 have been linked to chronic diseases such as asthma and allergic diseases such as rhinitis.⁴⁸ In our study, an inflammatory profile was observed in RAW 264.7 cells, with an increase in TNF levels, followed by a decrease in IL-10. A similar profile has been seen with other pesticides such as mancozeb, chlorothalonil, methyl thiophanate⁴⁹ and the pyrethroid permethrin.⁵⁰

IL-1β is a pro-inflammatory cytokine that affects all organs. An increased production is associated with numerous physiological changes, as it acts as a master regulator of the immune response.⁵¹ IL-1β activates several immune responses, such as the recruitment of neutrophils and the activation of chemokines and cytokines that stimulate adaptive immunity. It is normally secreted in response to the recognition of molecular patterns associated with pathogens. Chemicals that induce the secretion of IL-1ß may have the ability to induce “sterile inflammation,” an inflammation that occurs in the absence of microorganisms.⁵² Here, the levels of IL-1ß were increased in 3T3 cells, a pattern that was also observed for pesticides such as pentachlorophenol and dichlorodiphenyltrichloroethane (DDT).⁵³ Interestingly, studies in populations exposed to DDT associated the alteration of IL-1β levels with the development of cancer.^54,55

DNA damage is strongly linked to the toxicity of several pesticides. Due to the high levels of reactive substances, pesticides can interact with DNA and compromise its structure, in turn leading to the carcinogenesis processes.⁵⁶ Here, it was demonstrated for the first time that flumetralin increased DNA damage in both comet assay and LMW-DNA assay in 3T3 cells. This could be a consequence of flumetralin-induced cytotoxicity rather than a direct genotoxic effect, as observed by Cestonaro et al. (2023)¹⁶ with the insecticide imidacloprid.

Finally, our results demonstrate that flumetralin affects cell viability in different in vitro models at environmentally relevant concentrations. This growth-inhibitor interferes with the immune response, affecting the anti-inflammatory response of macrophages leading to immunosuppression. The immune system is essential to protect humans from several pathologies and this immune modulation could result in increasing the risk of allergic processes⁵⁷ and autoimmune diseases⁵⁸ in non-target species, including humans. These observations are backed up by our in silico predictions. Therefore, additional studies are necessary to assess the exposure of flumetralin with the development of pathologies using multicellular organisms.

Conclusion

In summary, flumetralin presented cytotoxicity to RAW 264.7 macrophages and 3T3 fibroblasts, producing reactive species and altering mitochondrial membrane potential. An inflammatory cytokines profile was observed in both cells, with increased levels of TNF and IL-1ß, and decreased levels of IL-10. Our study contributes to understanding the toxic effects of growth- inhibitor flumetralin and the risks associated to the exposure of non-target species.

Author contributions

Aline Mocellin Conte: Data curation; Formal analysis; Investigation; Writing—original draft. Fernanda Mocellin Conte: Formal analysis; Investigation; Writing—original draft. Larissa V. Cestonaro: Investigation. Maria Fernanda Nunes Ribeiro: Investigation. Rodrigo F. Silva: Investigation. Renata De Faveri: Investigation. Larissa Benvenutti: Investigation. Solange C. Garcia: Resources; Conceptualization. José Roberto Santin: Resources; Supervision, Writing—review & editing. Rodrigo Ligabue-Braun: Formal analysis; Writing—review & editing. Bruno Dutra Arbo: Resources; Supervision, Writing—review & editing. Marcelo Dutra Arbo: Resources; Conceptualization; Supervision, Writing—review & editing.

Funding

This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES Foundation, financial code 001).

Conflicts of interest. The authors declare that there is no conflict of interest.