# Immunotoxicity of relevant mixtures of pesticides and metabolites on THP-1 cells

Aliciane De Almeida Roque, Francisco Filipak Neto, Claudia Cosio, Iris

Barjhoux, Ciro Alberto De Oliveira Ribeiro, Damien Rioult

► To cite this version:

Aliciane De Almeida Roque, Francisco Filipak Neto, Claudia Cosio, Iris Barjhoux, Ciro Alberto De Oliveira Ribeiro, et al.. Immunotoxicity of relevant mixtures of pesticides and metabolites on THP-1 cells. Toxicology, 2023, 493, pp.153557. 10.1016/j.tox.2023.153557. hal-04382625

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Distributed under a Creative Commons Attribution 4.0 International License
IMMUNOTOXICITY OF RELEVANT MIXTURES OF PESTICIDES AND
METABOLITES ON THP-1 CELLS

Aliciane De Almeida Roque¹*, Francisco Filipak Neto¹, Claudia Cosio², Iris Barjhoux², Ciro Alberto de Oliveira Ribeiro¹*, Damien Rioult²,³*

¹Federal University of Paraná, Department of Cell Biology, Laboratory of Cell Toxicology, PO Box: 19031, CEP: 81531-980, Curitiba, PR, Brazil.

²Université de Reims Champagne-Ardenne, UMR-I 02 INERIS-URCA-ULH SEBIO Stress Environnementaux et BIOsurveillance des milieux aquatiques, 51097 Reims, France.

³Université de Reims Champagne-Ardenne, Plateau Technique Mobile de Cytométrie Environnementale MOBICYTE – INERIS, 51097 Reims, France.

*Corresponding Authors.

E-mail addresses: alicianeroque@gmail.com, ciro@ufpr.br, damien.rioult@univ-reims.fr
Pesticides are used to combat agricultural pests but also trigger side effects on non-target organisms. Particularly, immune system dysregulation is a major concern due to the organism's increased vulnerability to diseases, including cancer development. Macrophages play essential roles in innate and adaptive immunity and can undergo classical (M1) or alternative (M2) activation. The M1 pro-inflammatory phenotype has an antitumor role, while M2 favors tumor promotion. Although previous studies have linked pesticide exposure to immune compromise, macrophage polarization is still poorly studied. Here, we investigated the effects of 72 h-long exposure to the mixture of four pesticides widely used in Brazil (glyphosate, 2,4-D, mancozeb, and atrazine), and their main metabolites (aminomethylphosphonic acid, 2,4-diclorophenol, ethylenethiourea, and desethylatrazine) on human leukemia monocytic THP-1 cell line at concentrations based on the Acceptable Daily Intake (ADI) value established in the country. The data revealed immunotoxicity related to impaired cell metabolism in all exposed groups, decreased cell attachment (Pes: $10^{-1}$; Met: $10^{-1}$; Mix: all concentrations), and disturbance in nitric oxide (NO) levels (Met: $10^{-1}$, $10^{1}$; Mix: all concentrations). The polarization of macrophages towards a more pro-tumor M2-like phenotype was also supported by decreased secretion of the pro-inflammatory cytokine TNF-$\alpha$ (Pes $10^{0}$, $10^{1}$) and increased IL-8 (Pes $10^{1}$). These outcomes alert about the risk of pesticide exposure in the Brazilian population.

**Keywords:** cytokines, M2 polarization, TAMs, immunosuppression.
# 1. INTRODUCTION

Pesticides are chemicals widely used to combat agricultural pests, including unwanted weeds, insects, and microorganisms (Weis et al., 2019). Due to indiscriminate use, pesticides and their metabolites have been spread into the environment (Mokarizadeh et al., 2015). Consequently, there is a growing concern about human exposure to these chemicals. For example, in Brazil, a country recognized worldwide for its importance in the food supply, the intensive use of pesticides (>500,000 tons per year) causes high levels of exposure, as each inhabitant consumes about 7 L of pesticides per year (Braga et al., 2020; Panis et al., 2022; Weis et al., 2019). The ongoing changes in the regulation of pesticides in Brazil, such as Bill 6,299/2002, may aggravate this situation, as it intends to facilitate and speed up the approval process for new pesticides (Moraes, 2019).

Currently, glyphosate, which has been either banned or restricted in at least 20 countries (Meftaul et al., 2020), is the best-selling pesticide in Brazil, followed by 2,4-D, mancozeb, and atrazine; the latter banned in the EU since 2004 (IBAMA, 2021; EUR-Lex, 2005). The immunotoxicity and immunomodulatory potential of these isolated pesticides have already been demonstrated as the disruption of pro-inflammatory and anti-inflammatory cytokine levels, oxidative stress, and decreased viability in different *in vitro* and *in vivo* models (Chang et al., 2021; Chen et al., 2014; Corsini et al., 2006; Hong et al., 2017; Kaioumova et al., 2001; Pinchuk et al., 2007; Wang et al., 2023; Weis et al., 2019).

However, as humans are exposed to mixtures of pesticides (Hernández et al., 2017) studies with isolated exposures have limitations to establish the risk
of exposure (Mesnage et al., 2015). Furthermore, it is known that for some
active ingredients, metabolites can be more toxic than parental compounds
(Gullino et al., 2010; Lemes et al., 2005). Particularly in the context of cancer,
the reported immune impairment due to pesticide exposure deserves attention,
as immune disorders contribute to poor patient prognosis and tumor promotion
(Lee and Choi, 2020). Thus, as macrophages are one of the most abundant
immune cell populations in the tumor microenvironment (Labani-Motlagh et al.,
2020), a better understanding of the role of pesticide mixture exposure in
macrophage polarization could help to clarify the risk of exposure in the human
population.

Macrophages are mononuclear cells of monocytic origin (Sirenko et al., 2019) that play an important role in immune surveillance and tissue homeostasis. According to the microenvironment, macrophages can be polarized into classically activated (M1) or alternatively activated (M2) (Zhou et al., 2020). M1 macrophages produce high levels of reactive oxygen and nitrogen species, as well as pro-inflammatory cytokines, such as tumor necrosis factor (TNF-α), interleukins (IL)-1β, IL-6, and IL-12 (Genin et al., 2015), which endow the cells with high bactericidal and tumoricidal capacity (Edin et al., 2012). M2 macrophages release high levels of the anti-inflammatory cytokine IL-10 and chemokines that promote angiogenesis, especially IL-8 (also known as CXC-chemokine ligand - CXCL8) and are immunosuppressive, favoring tissue remodeling, angiogenesis, and tumor promotion (Edin et al., 2012; Genin et al., 2015; Mantovani et al., 2008; Zhou et al., 2020).

In cancer, tumor-associated macrophages (TAMs), recruited to the tumor
microenvironment (Genin et al., 2015; Kaushik et al., 2016), predominantly
exhibit the M2-like anti-inflammatory phenotype (Cieslewicz et al., 2013; Wang et al., 2019). Due to the secretion of pro-tumor factors, such as epidermal growth factor (EGF), fibroblastic growth factor (FGF), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF) (Genin et al., 2015), and proteases like matrix metalloproteinases 2 and 9, TAMs promote angiogenesis, and favor the survival of malignant cells and metastasis spread, leading to a poor prognosis in cancer patients (Genin et al., 2015; Zheng et al., 2019; Zhou et al., 2020). Thus, there is great interest in identifying compounds that stimulate macrophage polarization reprogramming for anticancer immunotherapy (Wang et al., 2019).

The human acute monocytic leukemia cell line (THP-1) can be easily differentiated into M0 macrophage by using phorbol 12-myristate 13-acetate (PMA) and then polarized either in the M1 phenotype via incubation with lipopolysaccharide (LPS) and IFN-γ or in M2 with IL-4 and IL-13 (Bahrami et al., 2021; Genin et al., 2015). These THP-1-derived M1 and M2 macrophages present cytokine expression and secretion profiles similar to those of human primary macrophages (Genin et al., 2015; Qin, 2012), and are considerably easier to obtain. Thus, THP-1 macrophages have become widely used as an *in vitro* model to evaluate the immunomodulatory activity of substances (Abuarqoub et al., 2022; Huo et al., 2020; Sirenko et al., 2019).

In the present study, we investigated the immunomodulatory effect of the mixture of the four main active ingredients of pesticides used in Brazil, as well as their main metabolites, on the THP-1 cell line after 72 h exposure through the analyses of cell viability and cytotoxicity (MTT, resazurin, crystal violet, and neutral red assays), reactive oxygen species (ROS) and nitric oxide (NO) levels,
phagocytic activity and inflammatory cytokines levels (TNF-α, IL-6, IL-1β and IL-8).

## 2. MATERIAL AND METHODS

### 2.1 Chemicals and stock solutions

The active ingredients of glyphosate, 2,4-dichlorophenoxyacetic acid, mancozeb, and atrazine, as well as their main metabolites (aminomethylphosphonic acid, 2,4-diclorophenol, ethylenethiourea, and desethylatrazine), were purchased from Sigma-Aldrich (95-99% pure). The concentrations used in this study were determined according to the acceptable daily intake (ADI) of each parent compound defined by the Brazilian Health and Surveillance Agency (ANVISA, 2021), with the values originally expressed in mg kg⁻¹ of body weight day⁻¹ converted to µg mL⁻¹ for cell exposure (Table 1). Since the metabolites are not regulated by Brazilian legislation, a concentration equal to the parental compounds was tested. For clarity, the pesticides, metabolites, and pesticides + metabolites mixtures will be referred to hereafter as Pes, Met, and Mix. To prepare the stock solutions, the individual compounds were initially diluted in pure dimethylsulfoxide (DMSO) and phosphate-buffered saline (PBS), according to their solubility, and stored at -20 °C.

<table><caption>Table 1 – Characterization of the tested mixtures.</caption><thead><tr><th rowspan="3">Active ingredient</th><th rowspan="3">CAS No</th><th colspan="10">ADI-folds (µg mL<sup>-1</sup>)</th></tr><tr><th colspan="3">Pes</th><th colspan="3">Met</th><th colspan="3">Mix</th></tr><tr><th>10<sup>-1</sup></th><th>10<sup>0</sup></th><th>10<sup>1</sup></th><th>10<sup>-1</sup></th><th>10<sup>0</sup></th><th>10<sup>1</sup></th><th>10<sup>-1</sup></th><th>10<sup>0</sup></th><th>10<sup>1</sup></th></tr></thead><tbody><tr><td>Glyphosate</td><td>1071-83-6</td><td>0.05</td><td>0.5</td><td>5</td><td></td><td></td><td></td><td>0.05</td><td>0.5</td><td>5</td></tr><tr><td>2,4-D</td><td>94-75-7</td><td>0.001</td><td>0.01</td><td>0.1</td><td></td><td></td><td></td><td>0.001</td><td>0.01</td><td>0.1</td></tr><tr><td>Mancozeb</td><td>8018-01-7</td><td>0.003</td><td>0.03</td><td>0.3</td><td></td><td></td><td></td><td>0.003</td><td>0.03</td><td>0.3</td></tr><tr><td>Atrazine</td><td>1912-24-9</td><td>0.002</td><td>0.02</td><td>0.2</td><td></td><td></td><td></td><td>0.002</td><td>0.02</td><td>0.2</td></tr><tr><td>AMPA</td><td>1066-51-9</td><td></td><td></td><td></td><td>0.05</td><td>0.5</td><td>5</td><td>0.05</td><td>0.5</td><td>5</td></tr><tr><td>2,4-DCF</td><td>120-83-2</td><td></td><td></td><td></td><td>0.001</td><td>0.01</td><td>0.1</td><td>0.001</td><td>0.01</td><td>0.1</td></tr><tr><td>Ethylenethiourea</td><td>6190-65-4</td><td></td><td></td><td></td><td>0.003</td><td>0.03</td><td>0.3</td><td>0.003</td><td>0.03</td><td>0.3</td></tr><tr><td>Desethylatrazine</td><td>96-45-7</td><td></td><td></td><td></td><td>0.002</td><td>0.02</td><td>0.2</td><td>0.002</td><td>0.02</td><td>0.2</td></tr></tbody></table>
ADI: acceptable daily intake (mg kg⁻¹ of body weight day⁻¹) converted to µg mL⁻¹. CAS No: Chemical Abstracts Service Registry Number; Pes: Pesticide mixture; Met: Metabolite mixture; Mix: Pesticide + metabolite mixture. 2,4-D- 2,4-dichlorophenoxyacetic acid; AMPA-Aminomethylphosphonic acid; 2,4 DCF- 2,4-diclorophenol. Concentrations based on the ADI of each parent compound (µg mL⁻¹): 10⁻¹: 10-fold lower than the ADI; 10⁰: equals to the ADI; 10¹: 10-fold higher than the ADI.

## 2.2. Cell culture and exposure

The THP-1 cell line was cultured in Roswell Park Memorial Institute Medium (RPMI 1640) supplemented with 10% Fetal Bovine Serum (FBS) heat-inactivated, penicillin (100 U m⁻¹) and streptomycin (100 µg m⁻¹) and was maintained at 37 °C, in a humidified atmosphere with 5% of CO₂.

For exposure, THP-1 cells were seeded in the culture plates (96-well plates: 3x10⁴ cells/well; 24-well plates: 1.2x10⁵ cells/well) and immediately treated with PMA (150 nM) to induce the differentiation into macrophages (Genin et al., 2015) for 24 h. Then, the culture medium was replaced by a fresh pre-heated medium containing the tested mixtures (PBS and DMSO for the control), and the cells were exposed for 72 h in the CO₂ incubator.

To prepare the culture medium for cell exposure, 1000x concentrate stock solutions were thawed and diluted in RPMI 1640 medium containing the antibiotics and PBS and, the DMSO concentration was adjusted to 0.05% for all experimental groups, including the control.

## 2.3 Flow cytometry analysis

THP-1 cells were seeded and cultured in 24-well plates (1.2x10⁵ cells/well). After exposure, the cytometric analyses were performed using an Accuri™ C6 flow cytometer (BD Bioscience) with 20,000 gated events in the THP-1 cell region.
### 2.3.1 Phagocytic activity and cell mortality

After 72 h exposure, the cells were incubated with fluorescent microspheres (100/cell) for 3 h at 37 °C, washed with PBS, and trypsinized (0.25% trypsin and 0.02% ethylenediaminetetraacetic acid - EDTA) for 5 min. Subsequently, the cells were resuspended in RPMI 1640 medium with 1% propidium iodide (PI) and immediately analyzed in the flow cytometer (Barjhoux et al., 2020). A positive control was added to each series of analyses and consisted of incubating the cells for 30 min with 20% ethanol. To analyze the results, cell mortality was expressed as the percentage (%) of PI+ cells and phagocytic activity as the % of cells that engulfed at least one fluorescent microsphere.

### 2.3.2 Reactive oxygen species (ROS)

ROS levels produced by phagocytic cells are frequently monitored due to their important role in immunity mechanisms during infectious processes, promoting microbicidal activity by damage to the microbial cell wall or genetic instability (Gonzalez Segura et al., 2020). The intracellular ROS detection was performed using the non-fluorescent dye dihydrorhodamine 123 (DHR) which, in the presence of ROS such as hydrogen peroxide ($H_2O_2$) and peroxynitrite ($ONOO^-$), is oxidized to fluorescent rhodamine (R123) (Sigma-Aldrich, 2022). After 72 h exposure, the cells were incubated with DHR123 (5 µM in RPMI 1640 medium) for 30 min at 37 °C, washed with PBS, trypsinized for 5 min, and resuspended in RPMI 1640 for immediate analysis in the flow cytometer. For the positive control, the cells were incubated with 200 µM tert-butyl hydroperoxide (TBH) in RPMI 1640 medium for 1 h before the incubation with
DHR123. To analyze the results, the median values of fluorescence captured
were exported and applied for comparison purposes.

### 2.3.3 Inflammatory cytokines

After 72 h exposure, the culture medium was collected, centrifuged at
1800 rpm for 3 min, and stored at -80 °C. The BD Cytometric Bead Array (CBA)
Human Inflammatory Cytokines Kit (Catalog No. 551811) was used to measure
protein levels of IL-12p70, TNF-α, IL-10, IL-6, IL-1β and IL-8 (pg mL⁻¹) in the
culture supernatants, according to the manufacturer's instructions.

## 2.4 Fluorescence and colorimetric analysis

THP-1 cells were seeded and cultured in 96-well plates (3x10⁴ cells/well). After exposure, the analyses were performed using the TECAN Infinite 200 microplate reader. Results were expressed as the mean value of replicates by each independent experiment relative to the negative control group.

### 2.4.1 Nitric oxide (NO)

After 72 h exposure, 150 µL of the medium was transferred to a black microplate, followed by the addition of 10 µL of 2,3-diaminonaphthalene (DAN, 0.5 mg ml⁻¹) in hydrochloride acid (HCl, 0.62 M). Under acidic conditions, DAN reacts with nitrite and generates the fluorescent product, 1-H- naphthotriazole (Misko et al., 1993). After 30 min of incubation (at room temperature protected from light), 5 µL of sodium hydroxide (NaOH, 2.8 M) was added to stop the reaction and intensify the fluorescent signal. Formation of 1-H- naphthotriazole was determined by fluorescence measurement (Ex/Em: 360/460 nm) (da Luz et
al., 2020). For the positive control, the cells were incubated with LPS (50 pg mL⁻¹ in RPMI 1640) for 24 h before the analysis (Sirenko et al., 2019).

### 2.4.2 Cell viability and cytotoxicity activity

**MTT:** after exposure, the cells were incubated in fresh RPMI 1640 medium containing 0.5 mg mL⁻¹ of MTT at 37 °C. After 1 h, the cells were washed twice with PBS, the formazan crystals were solubilized with pure DMSO, and the absorbance was measured at 560 nm (Mosmann, 1983).

**Resazurin:** the cells were concomitantly incubated with the tested mixtures and 44 µM of resazurin, and the absorbances of the oxidized (600 nm) and reduced forms (560 nm) of resazurin were measured at 0, 24, 48, and 72 h. The reduction percentage was calculated as described by Lee & Jain (2017).

**Neutral red:** after exposure, cells were incubated at 37 °C for 2 h in fresh RPMI 1640 medium containing 50 µg mL⁻¹ of neutral red. Then the cells were washed twice with PBS, the dye was extracted with a solution of 50% ethanol and 1% glacial acetic acid, and the absorbance was measured at 560 nm (Repetto et al., 2008).

**Crystal Violet:** after exposure, cells were dyed with crystal violet (0.25 mg mL⁻¹ in water) for 10 min at room temperature, washed twice with PBS, and the dye extracted with 33% glacial acetic acid for absorbance measurement at 560 nm (Kueng et al., 1989).

## 2.4 Data analysis

At least four independent experiments were performed for each endpoint. The normality of residuals and homoscedasticity were checked by the Shapiro-Wilk and Brown-Forsythe tests, respectively (α = 0.05). Parametric data were
analyzed by One-way ANOVA followed by Dunnett's *post hoc* test (α = 0.05) and expressed as the mean±95% confidence interval (C.I.). The nonparametric data were analyzed by Kruskal-Wallis non-parametric ANOVA followed by Dunn's multiple comparisons test (α = 0.05) and expressed as the median±interquartile range (25th and 75th percentiles). To provide a more comprehensive view of the result set, the data obtained through the 14 endpoints investigated in this study were scored and ranked according to the method described by Mbakidi et al. (2021). Briefly, this method scores the experimental groups according to statistical significance, ranking treatments from most to least toxic according to the calculated score.

### 3. RESULTS

The exposure caused a significant increase of NO levels in Met (10⁻¹ and 10¹: ↑7%), and all Mix concentrations (↑7-14%), however, prior incubation with LPS (50 pg mL⁻¹ in RPMI 1640 for 24 h) did not prove to be an adequate positive control in our study (Figure 1A). Pes, Met, and Mix exposures did not modulate the phagocytic activity of THP-1 cells (Figure 1B). Increases in cell mortality rate (↑394%), assessed by PI⁺ cells, and intracellular ROS production (↑679%) were observed in the positive controls only (Figures 1C-D).
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>Figure 1. Nitric oxide levels (A). Phagocytic activity (B). Cell mortality (P+). Reactive oxygen species (D). Ctrl: control (neg: negative/pos: positive); Pes: Pesticide mixture; Met: Metabolite mixture; Mix: Pesticide + metabolite mixture. 10⁻¹: 10-fold lower than the ADI; 10⁰: equals to the ADI; 10¹: 10-fold higher than the ADI; Rel. Ctrl: relative to the negative control; Flu: fluorescence; Eth: ethanol; TBHP: tert-butyl hydroperoxide; LPS: lipopolysaccharide. ANOVA + Dunnett's test or Kruskal-Wallis (KW) non-parametric ANOVA + Dunn's test. Dots: independent experiments; Horizontal bars: mean/median; vertical bars: confidence interval/interquartile range. Asterisks significant difference in comparison to the negative control with: \*p<0.05; \*\*p<0.01; \*\*\*p<0.0001. N = 4-6.</figcaption></figure>

The levels of TNF-α significantly decreased between 3.5 to 6.2-fold for Pes (10⁰ and 10¹), 2.9-fold for Met 10⁻¹, and 3.3-fold for Mix 10¹ (Figure 2A). The levels of IL-1β decreased 3.6-fold after exposure to the Met 10⁻¹ (Figure 2B), and IL-8 increased in a dose-dependent manner for the group Pes with
statistical significance for the highest concentration (↑1.7-fold, Figure 2C).

Conversely, the exposure to the pesticides and metabolites did not significantly affect IL-6 levels Figure 2D), and IL-12p70 and IL-10 levels were below the kit detection limit (20 pg mL⁻¹).

<figure><img src="image_6.png" /></figure>

Figure 2. Inflammatory cytokines levels (pg/mL): TNF-α (A). IL-1β (B). IL-8 (C). IL-6 (D). Pes: Pesticide mixture; Met: Metabolite mixture; Mix: Pesticide + metabolite mixture. 10⁻¹: 10-fold lower than the ADI; 10⁰: equals to the ADI; 10¹: 10-fold higher than the ADI. ANOVA + Dunnett's test or Kruskal-Wallis (KW) non-parametric ANOVA + Dunn's test. Dots: independent experiments; Horizontal bars: mean/median; vertical bars: confidence interval/interquartile range. Asterisks significant difference in comparison to the negative control with: \*p<0.05; \*\*p<0.01; \*\*\*p<0.001; \*\*\*\*p<0.0001. N = 5.
Regarding cell viability and cytotoxicity assays, the reduction percentage of resazurin was not modulated after 24 h exposure (Figure 3A) but increased after 48 h for Met (10⁻¹ and 10⁰: ↑15-20%, Figure 3B) and 72 h for Met 10⁻¹ and Mix 10⁻¹ (↑20-22%, Figure 3C). MTT metabolism significantly decreased after exposure to all mixture groups (↓23-37%, Figures 3D; S1A), and the number of attached cells, evaluated by the crystal violet assay, decreased after exposure to the lowest concentration of Pes (↓17%) and Met (↓18%) and all Mix concentrations (↓19-22%, Figures 3F; S1B). Conversely, no statistical significance was found related to neutral red retention after exposure to the tested mixtures (Figures 3E; S1C).
A % - ANOVA: p = 0.5840

<figure><img src="image_7.png" /></figure>

B % - ANOVA: p = 0.0026

<figure><img src="image_8.png" /></figure>

C % - KW: p = 0.0080

<figure><img src="image_9.png" /></figure>

D Rel. Ctrl - ANOVA: p = <0.0001

<figure><img src="image_10.png" /></figure>

E Rel. Ctrl - ANOVA: p = 0.0008

<figure><img src="image_11.png" /></figure>

F Rel. Ctrl - ANOVA: p = 0.3115

<figure><img src="image_12.png" /></figure>

Mixture based on ADI-folds

o Ctrl neg

● Pes

● Met

● Mix
Figure 3. Resazurin reduction percentage after 24 h (A). Resazurin reduction percentage after 48 h (B). Resazurin reduction percentage after 72 h (C). MTT metabolism (D). Crystal violet: attached cells (E). Neutral red retention(F). Ctrl: control; Pes: Pesticide mixture; Met: Metabolite mixture; Mix: Pesticide + metabolite mixture. 10⁻¹: 10-fold lower than the ADI; 10⁰: equals to the ADI; 10⁻¹: 10-fold higher than the ADI; Rel. Ctrl: relative control. ANOVA + Dunnett's test or Kruskal-Wallis (KW) non-parametric ANOVA + Dunn's test. Dots: independent experiments; Horizontal bars: mean/median; vertical bars: confidence interval/interquartile range. Asterisks significant difference in comparison to the negative control with: \*p<0.05; \*\*p<0.01; \*\*\*p<0.001; \*\*\*\*p<0.0001. N = 4-6.

Table 2 summarizes the scores and ranking of the 14 endpoints investigated in the present study. The set of results showed that, according to statistical significance, the lowest concentration of Met (#1) followed by Mix 10¹ (#2), Mix 10⁻¹ (#3), Mix 10⁰, and Pes 10¹ (#4) had the most prominent effects, while Pes 10⁰ (#5), Pes 10⁻¹, Met 10⁰ and 10¹ (#6) had the least pronounced effects on THP-1 cells.

<table><caption>Table 2 – Scores and ranks according to significant responses to tested mixtures.</caption><thead><tr><th>Group</th><th>NO</th><th>R48</th><th>R72</th><th>MTT</th><th>CV</th><th>TNF-α</th><th>IL-1β</th><th>IL-8</th><th>IL-6</th><th>Nb*</th><th>Scr%</th><th>Rk</th></tr></thead><tbody><tr><td>Pes 10<sup>-1</sup></td><td>ns</td><td>ns</td><td>ns</td><td>---</td><td>-</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>5</td><td>8.93</td><td>6</td></tr><tr><td>Pes 10<sup>0</sup></td><td>ns</td><td>ns</td><td>ns</td><td>---</td><td>ns</td><td>---</td><td>ns</td><td>ns</td><td>ns</td><td>7</td><td>12.5</td><td>5</td></tr><tr><td>Pes 10<sup>1</sup></td><td>ns</td><td>ns</td><td>ns</td><td>---</td><td>ns</td><td>---</td><td>ns</td><td>-</td><td>ns</td><td>8</td><td>14.29</td><td>4</td></tr><tr><td>Met 10<sup>-1</sup></td><td>+</td><td>++</td><td>+</td><td>---</td><td>-</td><td>-</td><td>-</td><td>ns</td><td>ns</td><td>12</td><td>21.43</td><td>1</td></tr><tr><td>Met 10<sup>0</sup></td><td>ns</td><td>+</td><td>ns</td><td>---</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>5</td><td>8.93</td><td>6</td></tr><tr><td>Met 10<sup>1</sup></td><td>+</td><td>ns</td><td>ns</td><td>---</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>5</td><td>8.93</td><td>6</td></tr><tr><td>Mix 10<sup>-1</sup></td><td>+</td><td>+</td><td>+</td><td>---</td><td>-</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>9</td><td>16.07</td><td>3</td></tr><tr><td>Mix 10<sup>0</sup></td><td>++</td><td>ns</td><td>ns</td><td>---</td><td>-</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>8</td><td>14.29</td><td>4</td></tr><tr><td>Mix 10<sup>1</sup></td><td>++</td><td>ns</td><td>ns</td><td>---</td><td>-</td><td>---</td><td>ns</td><td>ns</td><td>ns</td><td>11</td><td>19.64</td><td>2</td></tr></tbody></table>

Note: Pes: Pesticide mixture; Met: Metabolite mixture; Mix: Pesticide + metabolite mixture. 10⁻¹: 10-fold lower than the ADI; 10⁰: equals to the ADI; 10¹: 10-fold higher than the ADI; ns: not significant. NO: nitric oxide; R48: resazurin at 48 h of exposure; R72: resazurin at 72 h of exposure; CV: Crystal violet, IL: interleukin. Nb*: total number of asterisks per treatment corresponding to the sum of asterisks for each biological response (induction or inhibition) according to the p-value compared with the control (see Figures 1-3). Scr: scoring for each treatment expressed as a percentage by dividing the total number of asterisks for each parameter by the maximal possible number of asterisks per treatment (i.e., 14 endpoints x 4 asterisks = 56 → for Pes 10⁻¹, 5/56 = 8.93). Rk: treatment ranking from the most (#1) to the less toxic (#6) according to the calculated scoring. Asterisks were replaced by '+' and '-' symbols for upregulation and downregulation responses, respectively. Although not shown in the table, non-significant endpoints were also considered in the score calculation, and the details are given in Table S1.
4. DISCUSSION

Cytokines are secreted proteins that coordinate immune responses, including cell growth, differentiation, activation, and death of target cells (Borish and Steinke, 2003). The type of cytokine determines the nature of the immune responses, which can be cytotoxic, humoral, cell-mediated, or allergic (Borish and Steinke, 2003). Among the cytokines investigated by the present study, TNF-α was the most modulated by the tested mixtures. TNF-α is one of the main pro-inflammatory cytokines, produced by host cells in response to foreign agents or tumor cells, and acts on T (CD8+) and natural killer cells, leading to increased inflammation and tumor necrosis (Balkwill, 2009). The modulation of TNF-α by isolated exposure to some of the tested pesticides has been demonstrated by previous studies. However, as the results of these previous studies are quite varied, it is difficult to suggest any additive, synergistic or antagonistic effect of the compounds in the tested mixtures.

The decrease in TNF-α levels was also described by Corsini et al. (2005) in agricultural workers occupationally exposed to mancozeb, and later demonstrated by the downregulation of nuclear factor-KB (NF-KB) activation and blockage of TNF-α gene transcription in THP-1 cells exposed to mancozeb (5 µg mL⁻¹ for 1 h) (Corsini et al., 2006). Similar effects on TNF-α levels were found in serum and tumor tissue homogenate of BALB/C mice implanted with 4T1 cells subcutaneously and treated with atrazine (100 mg kg⁻¹ for 28 days) (Wang et al., 2023) and in peritoneal macrophages from mice exposed to the organophosphate chlorpyrifos (1 µM for 24 h) (Helali et al., 2016). Conversely, TNF-α levels increased in tissues of fish exposed via water to glyphosate (52.08 to 104.14 mg L⁻¹ for 24 to 168 h) (Ma and Li, 2015) and atrazine (4.28, 42.8, and
428 µg L⁻¹ for 40 days) (Chen et al., 2014), as well as in RAW 264.7 cells exposed to mancozeb (1 µg mL⁻¹ for 72 h) (Weis et al., 2019).

IL-1β is a key molecule of host defense against infection and injury (Lopez-Castejon and Brough, 2011) as it mediates the expression of genes that attract and activate cells of the adaptive immune system (Weber et al., 2010), or favor tumor promotion (Pan et al., 2020). Indeed, the IL-1β expression was previously reported in both M1 (Genin et al., 2015) and M2 (Pan et al., 2020) macrophages. In the current study, a decrease in IL-1β levels was observed only in cells exposed to the lowest concentration of the metabolite mixture. Different findings were observed by Pandey et al. (2019) and Tang et al. (2020) in rats orally exposed to glyphosate (100 and 250 mg kg⁻¹ for 14 days, and 50 and 500 mg kg⁻¹ for 35 days, respectively) and by Weis et al., (2019) in the supernatant of RAW 264.7 cells exposed to mancozeb (1 µg mL⁻¹ for 72 h). Unfortunately, we did not find studies in the literature reporting the effects of the metabolites used here for a deeper discussion.

Interestingly, IL-8 secretion increased significantly after exposure to the highest concentration of the pesticides and metabolites mixture, but this finding was not observed in cells exposed to the same concentration of the pesticide or metabolite mixtures which suggests antagonistic events among pesticides and metabolites. This kind of opposite action between pesticide mixtures was recently described by Abdelghani et al. (2019) and Lozano et al. (2018). The reduction of the predictive effect occurs when a compound stimulates the metabolism or interferes with the uptake (Hernández et al., 2013) or also by binding to different receptors with divergent effects (Hernández et al., 2017). According to Chen et al. (2014) and Ma et al. (2019), the increase of IL-8 levels
had also been described in common carp after waterborne exposure to atrazine
(4.28, 42.8, and 428 µg L⁻¹ for 40 days) and glyphosate (52.08 to 104 mg L⁻¹ for
1, 3 and 7 days), respectively. The increased IL-8 levels are a potent promoting
factor in many human cancers associated with angiogenesis and epithelial-
mesenchymal transition (EMT). As described by Cui et al. (2022), Fernando et
al. (2011), and Mantovani et al. (2008) high levels of IL-8 lead to increased
invasiveness and metastatic spread, corroborating the hypothesis that pesticide
mixtures may have a negative effect on cancer prognosis.

Together with the previous reports for isolated compounds, our findings regarding cytokine modulation by pesticide exposure highlight the complexity of studying mixtures. According to Diepens et al. (2017), the results observed for pesticide mixtures may vary according to the concentrations, components of the mixtures, exposure time, and endpoints investigated. However, due to the environmental relevance of risk assessment using complex mixtures, this approach should be encouraged. In this sense, the present study is one of the first to address immunomodulatory activities in human macrophages exposed to complex mixtures of pesticides and metabolites widely used in Brazil.

The modulation of these important cytokines, particularly the consistent
reduction in TNF-α secretion observed for all groups of cells, suggests that
mixtures of pesticides may induce the polarization of THP-1 macrophages
towards a more pro-tumor M2-like phenotype. Nevertheless, these findings
must be further investigated using specific markers, such as CD80 receptors for
M1 phenotype macrophages and CD206 and CD163 receptors for M2
phenotype macrophages (Genin et al., 2015).
The pro-tumor effect of M2 macrophages was evidenced by Genin et al. (2015) through co-incubation of HepG2 and A549 cell lines with M2 polarized macrophages, which highly protected these cancer cells from etoposide-induced apoptosis by decreasing the levels of cleaved caspase-3 and PARP-1 proteins. In contrast, co-incubation with M1 macrophages increased caspase activity and cancer cell death (Genin et al., 2015). Furthermore, increased M2 macrophage infiltration was associated with worse pathological features and poor prognosis in patients with prostate cancer (Comito et al., 2014).

According to the score ranking, cells exposed to the lowest concentration of Met showed the most pronounced effects based on statistical significance. The second and third positions were occupied by mixtures of pesticides + metabolites, which may suggest a cumulative toxic effect of these mixtures. Although ranked as least toxic concerning the overall score, the pesticide mixture groups had the most pronounced effects on modulating inflammatory cytokines, where decreased TNF-α levels (Pes 10⁰ and 10¹) and increased IL-8 (Pes 10¹) supported the hypothesis of macrophage immunomodulation triggered by these pesticides. These results certainly have an important meaning for human exposure risk since the concentrations of the mixtures used here were based on the ADI values established by Brazilian legislation for parental pesticides (ANVISA, 2021).

One of the main criticisms of ADI is the establishment of values based only on the isolated effects of active ingredients without considering mixtures (Mesnage et al., 2015; Perez-Carreon et al., 2009). For example, according to the latest report by the Brazilian Program for the Analysis of Pesticides Residues (PARA), 51% of the analyzed samples presented pesticide residues,
while 23% were considered unsatisfactory in terms of the Maximum Residue
Limit (MRL) (ANVISA, 2019). The concentrations tested in the present study
comprise values similar to and even lower than those already found in food and
human tissues by previous studies carried out in the country. According to
Candiotto et al. (2021), were detected alarming concentrations of glyphosate
and its metabolite AMPA in the urine of Brazilian farmers, ranging from 6,000 to
624,327 ppb, which means values 125-fold higher than the highest tested
concentration in the current study, in addition to concentrations of 2,4-D from
7.4 to 78.9 ppb (Candiotto et al., 2021). Mancozeb was detected in papaya (0.5
to 2.1 µg mL⁻¹) (Lemes et al., 2005), while its metabolite ethylenethiourea (ETU)
was found in the urine of greenhouse workers (171.6 µg g⁻¹ creatinine) (Costa
et al., 2022), showing that primary molecules and their metabolites can be
found together, highlighting the environmental relevance of the present findings.

A slight increase in NO levels was observed in the Met (10⁻¹ and 10¹) and
Mix (all concentrations) groups. According to a number of factors, such as
concentration, duration of exposure, cell sensitivity, and extracellular conditions,
NO can act both in inhibiting or promoting cancer (Kashfi et al., 2021). In terms
of tumorigenesis, NO contributes to tumor growth and metastatic spread via
increased glycolytic activity and regulation of cancer cell metabolism by
stimulating the Warburg effect (Kashfi et al., 2021). Therefore, the increase in
NO levels observed in these groups corroborates the hypothesis that exposure
to these substances may favor mechanisms associated with tumor promotion.

On the other hand, the absence of significant effects on intracellular ROS
levels may indicate the non-induction of redox imbalance in THP-1 cells by the
tested mixtures or be due to a detection limit of the DHR123 probe since
several authors have described its relative selectivity for ONOO⁻ over other forms of oxidants, such as H₂O₂, NO, and superoxide (O₂⁻·) (Dikalov and Harrison, 2014; Nugroho et al., 2015; Strijdom et al., 2006).

A reduced number of adherent cells and a decrease in MTT metabolism were found after exposure to the tested mixtures (Pes, Met, and Mix) without inducing cell death or reducing neutral red retention and resazurin reduction. These findings reveal the interference in cell adhesion mechanisms and the activity of dehydrogenases and diaphorases (Uzarski et al., 2017). The negative impact of pesticide exposure on immune cell viability has already been described by previous studies with some of these isolated compounds (Barbasz et al., 2020; Lee and Choi, 2020; Weis et al., 2019).

Together the combined reduction of TNF-α and increased levels of IL-8 found in the highest concentration of pesticide mixture suggest that exposure to the mixtures these compounds widely used in Brazil may stimulate the polarization of THP-1 macrophages towards a more pro-tumor state, like M2 phenotype. Therefore, it is likely that exposure to these mixtures may increase vulnerability to immune disorders and tumor promotion (Genin et al., 2015; Zhou et al., 2020). Furthermore, the disruption of NO levels accompanied by the effects on MTT and resazurin metabolism and the decreased number of adhered cells reveal the immunotoxicity of these compounds on THP-1 macrophages. This set of results supports the hypothesis of the immunomodulatory potential of these relevant mixtures of pesticides and metabolites and draws attention to the negative impacts that intensive use of pesticides may have on the human immune system and potentially on the prognosis of cancer.
**5. CONCLUSIONS**

The exposure to mixtures of widely used pesticides in Brazil and their most relevant metabolites caused immunotoxicity on human THP-1 cells, as demonstrated by the impaired cell metabolism in all exposed groups, decreased cell adhesion (Pes: $10^{-1}$; Met: $10^{-1}$; Mix: all groups) and increased nitric oxide (NO) production (Met: $10^{-1}$, $10^{1}$; Mix: all groups). Furthermore, data here linked pesticide mixture exposure and macrophage polarization to M2 phenotype due to the decreased secretion of the pro-inflammatory cytokines TNF-$\alpha$ (Pes $10^{0}$, $10^{1}$) and increased secretion of IL-8 (Pes $10^{1}$), which may favor tumor promotion. These findings are relevant since the investigated mixtures were composed of concentrations defined as safe by Brazilian legislation for isolated parental pesticides, and similar to those already found in food and humans for glyphosate, 2,4-D, and mancozeb, and the metabolites (AMPA and ethylenethiourea).

**Declaration of competing interest**

The authors have no conflict of interest to declare.

**Funding**

This study was supported by the Brazilian agencies National Council for Scientific and Technological Development (CNPq, finance code 428830/2018-8) and Coordination for the Improvement of Higher Education Personnel (CAPES, finance code 001), UMR-I 02 SEBIO laboratory and MOBICYTE core facility (France).
**Authors' contributions**

**Aliciane De Almeida Roque** participated in the development of the experimental design and research goals. Conducted the experiments, carried out the assays, processed and analyzed data with help from other authors, and wrote the original draft of the manuscript.

**Francisco Filipak Neto** participated with ideas in the experimental design plan and formulation of research goals, co-supervision the work, and worked on the original draft with the critical review, commentary, and revision.

**Iris Barjhoux** participated in the development of the experimental design, supervised the statistical data analysis and integration, and worked on the original draft with the critical review, commentary, and revision.

**Claudia Cosio** provided the study resources necessary for the experimental execution, like laboratory facilities, and worked on the original draft with the critical review, commentary, and revision.

**Ciro Alberto de Oliveira Ribeiro** conceived the idea for the project and formulated the research goals, provided part of the reagents, supervised the work, and worked on the original draft with the critical review, commentary, and revision.

**Damien Rioult** participated in the development of the experimental design and research goals and provided the study resources necessary for the experimental execution, like materials, reagents, and laboratory facilities. Also provided the instrumentation and computing resources for cytometric analysis,
supervised the experimental execution and data analysis, and worked on the
original draft with the critical review, commentary, and revision.

## Acknowledgments

We thank CAPES/PrInt/UFPR/RESMA, CNPq, the Graduate Program in Cell and Molecular Biology at UFPR, SEBIO – URCA Laboratory, and Mobicyte platform for financial support and for making this research possible. We also thank Pascal Maurice, the UMR-CNRS Medyc, Jessica Zablocki da Luz, Maristela Azevedo Linhares, and Newton Carlos Will for technical and logistic support.

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## Mixtures of the main pesticides and metabolites used in Brazil

<table><thead><tr><th>Pesticides</th><th>Metabolites</th></tr></thead><tbody><tr><td>• Glyphosate</td><td>• AMPA</td></tr><tr><td>• 2,4-D</td><td>• 2,4-DCF</td></tr><tr><td>• Mancozeb</td><td>• Ethylenethiourea</td></tr><tr><td>• Atrazine</td><td>• Desethylatrazine</td></tr></tbody></table>

72 h-exposure based on ADI values

<figure><img src="image_13.png" /></figure>

The effects may impair the immune responses and facilitate tumor promotion.