








key: cord-336659-qddjqiw9
authors: Ramos, Jheneffer Sonara Aguiar; Pedroso, Thays Millena Alves; Godoy, Fernanda Ribeiro; Batista, Renata Elisa; de Almeida, Frankcione Borges; Francelin, Carolina; Ribeiro, Francis Lee; Rocha-Parise, Michelle; de Melo e Silva, Daniela
title: Multi-biomarker responses to pesticides in an agricultural population from Central Brazil
date: 2020-08-21
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.141893
sha: 
doc_id: 336659
cord_uid: qddjqiw9

Abstract We evaluated farmworkers exposed to pesticides and individuals with no history of occupational exposure to pesticides. It was performed the comet assay to evaluate DNA damage. The immunophenotyping of TCD4+ lymphocyte subpopulations in peripheral blood was performed by flow cytometry. The single nucleotide polymorphisms (SNPs) in PON1, XRCC1, IL6, IL6R, TNF-α, and MIR137 genes were evaluated by real-time PCR. The exposed group was composed mostly by males (69.44%), with direct exposure to pesticides (56%) and with an average age range of 46 ± 13.89 years, being that 58.3% of farmworkers directly exposed to pesticides and reported the full use of personal protective equipment (PPE). DNA damage was greater in the exposed group (p < 0.05), reinforced by the use of PPE to denote a lower degree of DNA damage (p = 0.002). In this context, in the exposed group, we demonstrated that the use of PPE, age, gender and intoxication events were the variables that most contributed to increase DNA damage (p < 0.0001). Besides, the exposed group showed a significant increase in the subpopulations of T lymphocytes CD3+ CD4+ (p = 0.04) and CD3+ CD4+ CD25+ (p < 0.0001). SNPs in the TNF-α (rs361525) gene presented a difference in the genotype distribution between the groups (p = 0.002). The genotype distribution of TNF-α (rs361525) was also positively correlated with the DNA damage of the exposed group (r = 0.19; p = 0.01), demonstrating a higher risk of DNA damage in the farmworkers presenting the A mutated allele. Our findings demonstrate that pesticides can exert various deleterious effects on human health by damaging the DNA as well as by influencing the immune system in the case of both direct or indirect exposure and these issues are associated to age, gender, intoxication and the nonuse of PPE.

Pesticides have been extensively used globally to increase crop production and quality through controlling pests and for vector-borne diseases. It is noteworthy that

Brazil is one country that most consume pesticides in the world (Marcelino et al. Nascimento et al. 2020; Paumgartten, 2020) . Only in 2019, 439 products were authorized, the most significant number in the last 10 years (Brazilian Agriculture Minister, Nascimento et al. 2020 ) and even with the COVID-19 pandemic, the Brazilian government released 150 new pesticides this year (until may). Thus, almost all individuals are exposed to relatively low doses of pesticides due to environmental contamination or intentional use. According to Damalas and Eleftherohorinos (2011) , at low doses of exposure pesticides do not produce any permanent harmful effects on humans.

In this context, agricultural workers and their families and individuals who reside close to fields where pesticides are applied are considered to be the group that will receive the most considerable exposure at the highest risk for adverse health outcomes (Gangemi et al., 2016; Docea et al. 2017; Jacobsen-Pereira et al. 2018; Godoy et al. 2019; Marcelino et al. 2019) . As a result, humans exposed to a complex mixture of pesticides are more likely to develop different diseases due to deleterious effects on immune, hematological, nervous, endocrine, and reproductive systems (Corsini et al. 2013; Aroonvilairat et al., 2015; Campos et al. 2016; Corral et al. 2017; Docea et al. risk related to the use of pesticides and protect human health (Docea et al. 2017; Jacobsen-Pereira et al. 2018; Lozano-Paniagua et al. 2018 ; Barrón-Cuenca et al. 2019; Godoy et al. 2019) . The development and validation of new and useful biomarkers to assess pesticide exposure are warranted to implement proper control measures (Araoud, 2011; Lozano-Paniagua et al. 2018) .

Various in vitro and in vivo studies, as well as epidemiological approaches, have demonstrated that pesticides or their metabolites may result in genotoxic and mutagenic effects (Bolognesi, 2003; Docea et al., 2017; Kapeleka et al., 2019; Paumgartten et al., 2020) . Therefore, the use of genotoxicity and mutagenicity biomarkers is relevant to provide early identification of biological effects (Kapka-Skrzypczak et al., 2011; Aiassa, 2018; Lozano-Paniagua et al. 2018) . For assessment of mutagenic and genotoxic pesticide-induced damage, the most widely used methods are sister chromatid exchange assay, chromosomal aberrations test, single-cell gel electrophoresis (comet) assay, and micronuclei test (Aiassa, 2018; Godoy et al. 2019; Kapeleka et al., 2019; Marcelino et al., 2019) .

Data from experimental and epidemiological studies have also demonstrated that exposure to pesticides can modify the immune system either morphologically or functionally contributing to the development of immune-mediated diseases, such as asthma, allergies, type 1 diabetes, thyroid diseases, rheumatoid arthritis and atherosclerosis (Gangemi et al., 2016; Requena et al. 2019 ; Fukuyama and Tajiki-same author, in another study (Parks et al. 2019) , demonstrated that moderate to higher level of serum antinuclear autoantibodies are associated with the past exposure to some types of pesticides and a history of seeking medical care in male farmers occupationally exposed to pesticides.

In general, pesticides can impair immune cells function by inducing oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, disruption of the ubiquitin protease system or autophagy, and inhibition of enzymes with esterase activity (Corsini et al., 2008; Mokarizadeh et al., 2015; Fukuyama and Tajiki-Nishino, 2020) .

Therefore, the altered immune system may be a sensitive marker of pesticide-induced immunotoxicity, eventually affecting the development of immune-mediated disorders, and so may be predictive of eventual diseases (Corsini et al., 2013; Fukuyama and Tajiki-Nishino, 2020) . Biomarkers recommended assessing immunotoxicity of pesticides include lymphocyte count, antibody-mediated immunity (serum concentrations of immunoglobulins) analysis, lymphocytes phenotypic analysis by flow cytometry, measurements of autoantibodies and markers of an inflammatory response, among others (Rojas-García et al., 2011; Parks et al. 2019; Fukuyama and Tajiki-Nishino, 2020) .

It is also of particular relevance to the the environmental health research to investigate Single Nucleotide Polymorphisms (SNPs) in inflammatory genes since SNPs play a critical role in the assessment of the immune response to pesticide play a role in the risks of pesticide-related disease and can also be used as biomarkers associated with susceptibility (Gangemi et al., 2016; Sisto et al., 2019) .

Regarding the metabolism genes, SNPs in cytochrome P450 (CYP), glutathione transferases (including GSTM1, GSTP1, GSTT1), acetyltransferases (NAT2), and paraoxonases (mostly PON1) genes have been widely used to evaluate interindividual differences in metabolization and detoxification of pesticides (Rojas-García et al., 2011; Teodoro et al., 2019) . Additionally, polymorphisms in DNA repair genes, especially those involved in base excision repair, including OGG1 (8-oxoguanine DNA glycosylase) and XRCC1 (X-ray repair cross-complementation group 1) can be associated with higher risks of pesticide-related diseases. Moreover, gene variants have also been investigated to understand the differences in susceptibility to pesticide exposure (Tabrez et al., 2014; Teodoro et al., 2019) .

Hence, the current study evaluated the impact of pesticide exposure on the health of rural workers in the southeast and southwest of Goias, Brazil, using genotoxicity, immunotoxicity, and susceptibility tests. This is a pioneer study in Central

Brazil involving genetic and immunological biomarkers to identify how pesticides could impair such systems increasing the susceptibility to the development of chronic issues problems.

was composed of 100 individuals directly exposed to pesticides (occupationally exposed to various pesticides during storage, mixing, loading, and pesticide spraying activities), and 80 subjects indirectly exposed, living nearby crops, therefore, environmentally exposed to pesticides. The control group consisted of 180 individuals, 125 men, and 55 women, with no direct contact or closer exposure to pesticides, and were matched with the exposed group by age, gender, and lifestyle. It is worth mentioning that we sampled the total blood of the rural workers during the midseason and at the end of a week of application, once we could verify how exposure to such products altered the response of multiple biomarkers.

We evaluated DNA damage by alkaline single cell gel electrophoresis (comet assay) according to Singh et al. (1988) , with a slight modification, mainly in the stained of the slides. Briefly, two slides were processed for each individual. Ficoll-Paque™ Plus (density 1.077 g/mL, GE Healthcare, Uppsala, Sweden) following the manufacturer's instructions. Briefly, ten milliliters of well-mixed whole blood were for each sample were acquired and analyzed.

Genomic DNA was extracted from whole peripheral blood using the The estimation of genotype and allele frequencies was carried out by direct counting. Allele and genotype frequencies were compared between pesticide-exposed J o u r n a l P r e -p r o o f individuals and controls by the χ2 test. We also tested Hardy-Weinberg Equilibrium (HWE). The Hardy-Weinberg equilibrium is considered the null model of Population

Genetics and serves to test hypotheses about the change in allele and genotype frequencies in populations. In an infinite population, in which matings occur at random and in the absence of evolutionary factors such as natural selection, mutation and gene flow, the allelic and genotype frequencies remain constant throughout the generations, and are, respectively, p and q and p 2 , 2pq and q 2 (Mayo, 2008). These analysis were carried out with Genepop v. 1.2 (Raymond and Rousset, 1995) . The p-value was between 1 and 5 percent (p < 0.01 or < 0.05, respectively).

The demographic characteristics of the study group are shown in Table 1. The exposed and control groups did not differ regarding age, gender, smoking habits, and alcohol consumption. The time of exposure of the exposed group presented a median of 16.3 ± 10.3 years. The use of PPE was described by 58.3% of the farmworkers, while 41.7% reported not using it at all. A total of 37 individuals of the exposed group (20.6%) reported acute pesticide poisoning consisting of 10 indirectly exposed subjects (27%) and 27 directly exposed individuals (73%). Of the 27 directly exposed rural workers who were intoxicated, six of them did not use PPE. One hundred (87 men and 13 women) were individuals directly exposed to pesticides and 80 subjects (38 men and 42 women) live nearby crops and, therefore, were environmentally exposed to pesticides.

The most frequently self-reported chronic health problems among exposed individuals were high blood pressure (18.9%), allergy (17.2%), diabetes type 2 (11.1%), J o u r n a l P r e -p r o o f Journal Pre-proof and thyroid disease (4.4%). Regarding pesticides and in accordance to the organism they kill (Megha et al, 2018), exposed individuals reported frequent use of herbicides (47%) and followed by insecticide (42%) and fungicide (11%). The more commonly used pesticides were glyphosate (40.86%), 2,4-D (15.59%), cypermethrin (10.22%), deltamethrin (8.06%), and atrazine (5.38%). The most common crops were soybean (48%) and corn (34%) crops.

DNA damage in exposed and controls are described in Table 2 . We demonstrated more DNA damage in the exposed group compared to the non-exposed group (p < 0.05), independent of the type of exposure (if direct or indirect) [ Table 2 ].

No significant difference in the DNA damage was observed based on smoking habits and alcohol consumption (p > 0.05). Age, gender distribution, the use of PPE and intoxication events showed statistically significant differences in DNA damage (p < 0.001), demonstrating considerable DNA lesions among older farmworkers, women, individuals that reported intoxication and who did not use PPE [ Table 3 ]. We did not find association between time of exposure to pesticides and DNA damage (p>0.05).

Of the 360 individuals, 173 were analyzed by flow cytometry (118 exposed and 55 non-exposed). The individuals in the exposed group showed a significant increase of CD3 + CD4 + (p <0.05) , CD3 + CD4 + CD25 + (p < 0.0001) and CD3 + CD4 + CD25 + FOXP3 + (p < 0.05) lymphocytes subpopulations when compared to individuals in the non-exposed group (Figure 2A,2B and 2C) . However, the percentage of natural regulatory T cells (CD3 + CD4 + CD25 + FoxP3 +) did not differ between the J o u r n a l P r e -p r o o f Journal Pre-proof groups (p = 0.12) ( Figure 2D ). There were no statistically significant correlations between the percentage of TCD4 + cells and TCD4 + CD25 + cells and the percentage of DNA in tail (% DNA) in the exposed group (r = -0.15; p = 0.25 and r = -0.14; p = 0.13, respectively) [ Figure 3 ].

The distributions of all genotypes were in accordance to Hardy-Weinberg equilibrium. The SNPs genotyping of PON1 (rs662), XRCC1 (rs25487), IL6

(rs1800795), IL6R (rs2228145), and MIR137 (rs1625579) revealed that there was no statistically significant difference in the genotype and allele distributions among the studied groups (p > 0.05) ( Table 4 ). We also did not find that the farmers presenting mutant alleles of PON1 (rs662), XRCC1 (rs25487), IL6 (rs1800795), IL6R

(rs2228145), and MIR137 (rs1625579) presented increased DNA damage (Figure 4 ).

However, there was a difference in the SNPs distribution of the TNF-α (rs361525) gene between the study groups (p = 0.002) [ Figure 4 ]. Additionally, a significant positive correlation was found between the TNF-α (rs361525) polymorphism and DNA damage (r = 0.19; p = 0.01), but we found no correlation between TNF-α (rs361525) and the percentage of DNA in tail (% DNA) in the control group (r = 0.05; p = 0.47).

This is the first study from Central Brazil that demonstrated how genetic and immune biomarkers are associated to the exposure of a complex mixture of pesticides Also, families of farmworkers are often environmentally exposed to multiple pesticides, either by living near crops or by having contact with contaminated clothes and work tools without personal protection (Damalas and Eleftherohorinos, 2011; Parks, 2016; Doğanlar et al. 2018 ). In general, farmworkers' families are exposed to lower levels but for a longer duration to pesticides. Thereby may be more vulnerable to adverse effects, especially pesticide poisoning (Ward et al., 2006; Shirangi et al., 2010; Parks, 2016) . The chronic diseases reported by pesticide-exposed participants in our study is in agreement with studies performed in similar conditions (directly and indirectly exposition) (Mrema et al., 2017; Kongtip et al., 2018; Barrón-Cuenca et al. 2019) . Moreover, other epidemiological studies have shown a higher risk of psychiatric problems, in people exposed to pesticidesespecially those who have suffered from Journal Pre-proof associated pesticide diseases. However, the statistical associations between exposure to certain pesticides and the incidence of some chronic diseases are compelling enough to create concern. Besides the chronic effects observed in individuals exposed to pesticides, we also observed an increase in the DNA damage of exposed individuals compared to the control group (p < 0.05). Previous studies on farmworkers exposed to In agreement with the gender of exposed individuals, women displayed higher levels of DNA damage than men (p < 0.001), probably because they are the most actively involved laborers in the crops until harvest, as reported by Rekhadevi et al. 2017 and are also responsible for washing their partners' working clothes or male relatives. Therefore, women had direct and chronic contact with pesticides and their residues. Indeed, comparing genotoxic damage in individuals who reported using full PPE relative to those who did not use PPE or used incomplete PPE demonstrated higher levels of DNA damage among the latter group (p < 0.001). We also found increased J o u r n a l P r e -p r o o f Journal Pre-proof DNA damage in older farmworkers and in those that reported intoxication. Such findings demonstrated an influence of PPE's effectiveness in preventing the genotoxic effects of pesticides on peripheral blood cells (Simoniello et al., 2008) . Previous studies have shown the same correlations between PPE use, intoxication, age and gender on level of DNA damage and suggested to be due to differences in exposure conditions, DNA repair capability and lifestyle factors (Simoniello et al., 2008; How et al., 2015; Ali et al. 2017; Cayir t al., 2019) . According to Ali et al. 2017, women from Bahawalpur District, of the Punjab province, in India, exposed to pesticides while picking cotton with bare hands, presented increased DNA damage when compared to controls. Besides, the DNA damage was positively correlated to age and exposure time, demonstrating that DNA repair capability could be committed.

On the other hand, our results did not show any influence of smoking habits and alcohol consumption on genotoxic damage. Therefore, increased DNA damage in the exposed group was due to exposure to pesticides, and not associated with other confounding factors. Similar results were also produced by other authors, who found no significant difference between lifestyle and DNA damage (Simoniello et al., 2008; Kaur et al., 2011; Wilhelm et al., 2015) , unlike Barron-Cuenca et al. (2019) and Hayat et al. (2019) , which showed an increase of DNA damage.

Moreover, CD4 + lymphocytes subpopulations were also analyzed as a biomarker of alterations on the immune system. Our analysis demonstrated significant modifications of immunotoxicity parameters in the exposed group, specifically in the percentages of TCD4 + , T CD4 + CD25 + and TCD4 + CD25 + FoxP3 + lymphocytes subsets that indicates that the pesticides evoke an alteration in the CD4 + lymphocytes in the peripheral blood of exposed individuals. However, the percentage of natural regulatory T cells CD3 + CD4 + CD25 + FoxP3 + and did not differ significantly between the exposed J o u r n a l P r e -p r o o f and the non exposed groups. Our findings are promising as we demonstrate a disturbance in the pattern of TCD4 + lymphocytes subpopulation in front of the pesticide and a maintenance of the natural regulatory T cells percentage. This data strongly suggests that the pesticides evoke a peripheral T cell subpopulation alteration, what is plausible to purpose, would commit further immune reactions. At this moment our group is working to better explore this data.

Another important issue is individual susceptibility that influences physiological responses to pesticide exposure. Therefore, it is essential to identify genotypes that determine the modulation of the proteins involved in the metabolization, detoxification, and DNA repair, influencing the heterogeneity of responses to pesticides (Oliveira et al., 2019) . Regarding susceptibility biomarkers, only TNF-α rs361525

polymorphism showed a significant difference in genotype distributions between exposed and control groups (p = 0.002). TNF-α gene encodes tumor necrosis factoralpha (TNF-α) proinflammatory cytokine that is enhanced by the oxidative stress pesticide-induced as reported by some authors (Mecdad et al., 2011; Gangemi et al., 2016) .

In this study, subjects exposed to a complex mixture of pesticides had a significantly positive correlation between the TNF-α (rs361525) polymorphism and the DNA damage (r = 0.19; p = 0.01). All together, these findings indicate that the higher prevalence of A allele constitutes a susceptibility factor for the DNA damage observed in the pesticide-exposed farmworkers. Finally, genotype and allele frequencies of PON1, XRCC1, IL6, IL6R and MIR-137 were similar to other studies (Wong et al., 2008; Singh et al., 2011; Satti et al., 2013; Mahmoudi and Cairns, 2017) and did not present distinct distribution between exposed and unexposed groups. (2), 102-190, DOI: 10.1080 102-190, DOI: 10. /19338244.2013 Intranuovo, G., Schiavulli, N., Cavone, D., Birtolo, F., Cocco, P., Vimercati, L., Macinagrossa, L., Giordano, A., Perrone, T., Ingravallo, G., Mazza, P., Strusi, M., Spinosa, C., Specchia, G., & Ferri, G. M. 2018. Assessment of DNA damages in lymphocytes of agricultural workers exposed to pesticides by comet assay in a crosssectional study. Biomarkers, 23(5), 462-473. https://doi- J o u r n a l P r e -p r o o f 

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This work received financial support from SUS Program (PPSUS/GO -

