e45

Universidade Federal de Santa Maria

Ci. e Nat., Santa Maria, v. 43, e45, 2021

DOI: 10.5902/2179460X44026

ISSN 2179460X

Submitted: 01/05/2020 • Approved: 19/10/2020 • Published: 18/06/2021

Biology-Ecology

Redox signaling and inflammatory statuses of farmers exposed to agrochemicals

Perfil redox e inflamatório de agricultores expostos a agrotóxicos

Roberta Cattaneo HornI

Mariana Spanamberg MayerII

Patrícia WolkmerIII

Ana Caroline TissianiIV

Aimê Cunha ArrudaV

Gabriela Bonfanti AzzolinVI

Paulo Ricardo MoreiraVII

Diego Pascoal GolleVIII

Tiago Antonio HeringerIX

Caroline AlegransiXI

Jana KoefenderX

I Doutora, em Ciências Biológicas, Bioquímica Toxicológica, Universidade de Cruz Alta, Cruz Alta, RS, Brazil

https://orcid.org/0000-0001-9258-8005 – robertacattaneo82@gmail.com

II Acadêmica de Biomedicina, Universidade de Cruz Alta, Cruz Alta, RS, Brazil

https://orcid.org/0000-0002-2514-3783 – mspanamberg@gmail.com

III Mestrado Profissional em Desenvolvimento Rural, Universidade de Cruz Alta, Cruz Alta, RS, Brazil

https://orcid.org/0000-0001-6474-0102 – pwolkmer@unicruz.edu.br

IV Mestranda em Bioexperimentação, Universidade de Cruz Alta, Cruz Alta, RS, Brazil

https://orcid.org/0000-0002-9715-2283 – ana.c.t@hotmail.com

V Mestranda em Atenção Integral à Saúde, Universidade de Cruz Alta, Cruz Alta, RS, Brazil

https://orcid.org/0000-0002-9715-2283 – aimecunha4@gmail.com

VI Doutora em Farmacologia, Universidade de Cruz Alta, Cruz Alta, RS, Brazil

https://orcid.org/0000-0003-2602-6092 – gbonfanti@unicruz.edu.br

VII Doutor em Medicina, Universidade de Cruz Alta, Cruz Alta, RS, Brazil

https://orcid.org/0000-0002-3001-1988 – pmoreira@unicruz.edu.br

VIII Doutor em Engenharia Florestal, Universidade de Cruz Alta, Cruz Alta, RS, Brazil

https://orcid.org/0000-0002-5264-8007 – antoniother408@gmail.com

IX Graduado em Biomedicina, Universidade de Cruz Alta, Cruz Alta, RS, Brazil

https://orcid.org/0000-0001-7024-7891 – ana.c.t@hotmail.com

X Acadêmica de Farmácia, Universidade de Cruz Alta, Cruz Alta, RS, Brazil

https://orcid.org/0000-0002-6632-6543 – calegransi@gmail.com

I Doutora em Agronomia, Universidade de Cruz Alta, Cruz Alta, RS, Brazil

https://orcid.org/0000-0002-5882-9669 – jkoefender@unicruz.edu.br

ABSTRACT

Physiological changes take place in the human body upon exposure to pesticides. These changes may be followed by oxidative stress, which is caused by imbalance between the antioxidant defense system and reactive oxygen species (ROS) generation. Pesticides can cause inflammatory changes, which alter the structure of some cytokines essential for the functioning of the human body. Therefore, the aim of the present study is to assess the oxidative and inflammatory status of farmers exposed to pesticides. Blood samples from 50 exposed farmers and 50 non-exposed individuals (Control Group - CG) were collected. Oxidative stress and inflammatory marker levels were assessed. Na increase in TBARS (p<0.0001), IL-6 (p=0.03700), IL-8 (p=0.0011) and IL-10 (p=0.0011) levels was found, a decrease in enzymatic activity: SOD (p<0.001), CAT (p=0.0251), GPx (p<0.001), GST (p<0.001), GR (p<0.0001) in the evaluted farmers in relation to the CG data. Farmers demonstrated high oxidative damage. Low levels of antioxidants and inflammatory changes.

Keywords: Farmers; Intoxication; Oxidative stress

RESUMO

Durante a exposição a agrotóxicos ocorrem alterações fisiológicas no organismo humano, como o estresse oxidativo, caracterizado por um desequilíbrio entre o sistema de defesa antioxidante e a geração de espécies reativas de oxigênio (EROs). Os pesticidas podem ocasionar alterações inflamatórias, em algumas citocinas, que são essenciais para o funcionamento do nosso organismo. Neste contexto, o objetivo desse estudo foi avaliar o perfil oxidativo e inflamatório de agricultores expostos a agrotóxicos. Foram utilizadas amostras de sangue de 50 agricultores e 50 indivíduos não expostos a agrotóxicos (Grupo Controle – GC). Foram mensurados no plasma dos participantes marcadores de dano oxidativo e marcadores inflamatórios. Foi encontrada uma elevação nos níveis de TBARS (p<0,0001), IL-6 (p=0,03700), IL-8 (p=0,0011) e IL-10 (p=0,0011), diminuição da atividade das enzimas: SOD (p<0,001), CAT (p=0,0251), GPx (p<0,001), GST (p<0,001), GR (p<0,0001), GSH (p<0,0001) e BChE (p<0,0001) nos agricultores avaliados em relação aos dados do GC. Os agricultores demonstraram um elevado dano oxidativo, baixos níveis de antioxidantes e alterações inflamatórias.

Palavras-chave: Produtores agrícolas; Intoxicação; Estresse oxidativo

1 Introduction

Pesticide use has been growing globally, as it has become essential for crop management and pest control. Rio Grande do Sul is among the four states where pesticide use is most widespread in Brazil (DE MORAES, 2019). Rural workers are directly exposed to pesticides, as these substances have several chemical properties with different biological functions and actions; therefore, they can be highly toxic to human health (JACOBSEN-PEREIRA et al., 2018). Thus, appropriate use of Personal Protective Equipment (PPE) by rural workers is essential to protect their health (RISTOW et al., 2020).

The human body undergoes significant physiological changes upon exposure to pesticides. Oxidative stress may result from it, given the imbalance between the antioxidant defense system and Reactive Oxygen Species (ROS) production (YARIBEYGI et al., 2018). Absorption of toxic substances by the biological system triggers ROS production. When these substances are not eliminated by the antioxidant defense system, the following issues may arise: Enzyme inactivation, excessive consumption of endogenous antioxidants (ANTONIJEVIC et al., 2018; CLASEN et al., 2018), cell damage such as oxidation of cellular molecules through Lipid Peroxidation (LPO) (AKBEL et al., 2018) protein carbonylation and excessive activation of pro-inflammatory genes. As a result, rural workers exposed to pesticides develop serious diseases (SILVERIO; PINHEIRO, 2020).

Cell damage suffered by farmers can be assessed through the study of oxidative stress and inflammatory markers, which helps understanding biological changes caused by exposure to agrochemicals (FARKHONDEH et al., 2020).

Since rural workers exposed to pesticides are more likely to suffer from physiological changes, high levels of oxidative biomarkers and changes in inflammatory markers, the aim of present study was to assess redox signaling and inflammatory status of farmers exposed to pesticides due to labor activities.

2 Methods

2.1 Ethical considerations

The present study is subproject of research projects known as “Occupational monitoring of rural workers exposed to agrochemicals based on biomarker using” approved by the Research Ethics Committee of Cruz Alta University (UNICRUZ) under Substantiated Opinion n. 0071.0.417.000-11. This is an analytical cross-sectional study on the biological material of rural workers exposed to pesticides due to labor activities. All participants signed the informed consent form.

2.2 Population and Sample

Farmers who participated in the study were chosen at random, but they met the inclusion criterion: having been exposed to pesticides at any degree. Collections were carried out in Alto Jacuí region, Rio Grande do Sul State, which is known for the presence of large rural properties. Fifty farmers exposed to pesticides expressed their wish to participate in the study. Samples were randomly collected from 50 individuals non-exposed to pesticides due to labor activities (control group); this number was equivalent to that of exposed farmers. The study included participants in the age group 18 to 60 years. Individuals with the following chronic diseases were excluded from the research: Asthma, high blood pressure, diabetes mellitus, among others. Therefore, the study encompassed 100 participants, in total.

All study participants (farmers and controls) answered a questionnaire composed of a structured set of questions regarding age, sex, farming duration, time since last exposure to pesticides, PPE using and most used pesticides.

2.3 Blood collection and sample preparation

Blood collection was carried out at participants’ homes. First, blood was collected into vacuum tubes pre-filled with ethylenediaminetetraacetic acid (EDTA) for plasma analysis. Next, blood samples were centrifuged at 3000 rpm for 10 minutes. The samples were stored in freezer at -20ºC for further laboratory analyses.

2.4 Laboratory analyses

All the collected plasma was analyzed in triplicate. Glutathione S-transferase (GST) enzyme activity was determined through the method described by Habig, Pabst and Jakoby (1974). Results were expressed in μmol GS-DNB/min/mg protein. CAT enzyme activity was determined based on the method described by Nelson (1972) and results were expressed in nmol/mg ptn/min. The activity of enzymes Glutathione Reductase (GR), Glutathione Peroxidase (GPx) and Superoxide Dismutase (SOD) was determined with the aid of VIDA biotecnologia® (commercial kit brand) in colorimetric microplate readers - results were expressed in pg/mL. Reduced Glutathione (GSH) levels were determined through the method described by Ellman (1959) - results were expressed in µmol GSH/mL.

Thiobarbituric Acid Reactive Substance (TBARS) levels were determined according to the protocol by Jentzsch et al., (1996). Results were expressed in ηmol/mL (MDA). Total protein (ptn) was measured with the aid of Labtest® (commercial kit) and Protein Carbonylation (PC) levels were measured according to the method described by Levine (1990). Results were expressed in ηmol/mg ptn .

Butyrylcholinesterase (BChE) enzyme activity was measured with the aid of Labtest® (commercial kit) and results were expressed in UI/mL. Interleukin (IL-6, IL-8, IL-10) levels and the Tumor Necrosis Factor (TNF-α) were determined through microplate-based screening with the aid of VIDA biotecnologia® (commercial kit brand) - results were expressed in pg/mL.

2.5 Statistical analyses

Results were expressed in mean ± standard deviation. Data normality was assessed through Kolmogorov-Smirnov and Shapiro-Wilk tests. The assessed groups were compared through nonparametric student’s t-test. Differences between groups were considered statistically significant when p<0.05.

3 Results and discussion

Fifty farmers exposed to agrochemicals for more than 8 years (exposed group) and 50 healthy individuals - not exposed to pesticides - (control group) participated in the study. Farmers’ mean age was 52 years and 92.62% of them were men, whereas control participants were mean age of 32 years and 62.15% of them were men. The questionnaire assigned to the participants has shown that the mean time of agrochemical exposure was 29 years and the last exposure happened at least 90 days before the assessment.

Results recorded for the PPE using assessment evidenced that 46.06% of farmers have used it, 11.09% have used it occasionally and 42.85% did not use any type of PPE. The most widespread pesticides belonged to the following categories: organophosphates, carbamates, pyrethroids, organochlorides, fungicides and herbicides. Exposed farmers had lower enzyme activities than the control group (Fig. 1): SOD (A) (p<0.001), CAT (B) (p=0.0251), GPx (C) (p<0.001), GST (D) (p<0.001) and GR (E) (p<0.0001). Moreover, farmers’ GSH levels were lower than those of individuals non-exposed to agrochemicals (p<0.0001) (Fig. 2).

Farmers reported increased TBARS biomarker levels (p<0.001) (Fig. 3). However, PC levels (p=0.3505) were not significantly different between the assessed groups (data not shown). The exposed group reported BChE enzyme activity (p<0.0001) lower than that of the control group (Fig. 4).

Furthermore, according to figure 5, the exposed group reported increased IL-6 (A) (p=0.03700), IL-8 (B) (p=0.0011) and IL-10 (C) (p=0.0011) levels. There was no significant difference in TNF-α levels among farmers (p=0.4620) (data not shown).

Figure 1 – Antioxidant enzyme levels in the plasma of farmers occupationally exposed to pesticides

(A) Superoxide Dismutase (SOD); (B) Catalase (CAT); (C) Glutathione Peroxidase (GPx); (D) Glutathione-S-Transferase (GST); (E) Glutathione Reductase (GR)