Oxidative stress and renal status of farmers exposed to pesticides in Seville (Spain)

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in: Science of the Total Environment on 2024, available at: https://doi.org/10.1016/j.scitotenv.2024.175180” 1 Oxidative stress and renal status of farmers exposed to 1 pesticides in Seville (Spain) 2 Alfredo G. Casanova1, María G. Hinojosa2,*, Carmen Chamorro-López3, José Martín-Reina3, Raúl 3 Aguilera-Velázquez4 , Juan D. Bautista4, Ana I. Morales1#, and Isabel M. Moreno3# 4 1 Toxicology Unit, Universidad de Salamanca; Institute of Biomedical Research of Salamanca (IBSAL); Group of 5 Translational Research on Renal and Cardiovascular Diseases (TRECARD), Salamanca, Spain 6 2 Toxicology Unit, Department of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden 7 3 Department of Nutrition and Bromatology, Toxicology, and Legal Medicine, Faculty of Pharmacy, University of 8 Sevilla, Sevilla, Spain 9 4 Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain 10 11 12 # These authors share senior authorship. 13 * Corresponding author: María G Hinojosa. Toxicology Unit, Department of 14 Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Mail: 15 [email protected] 16 17 18 19 20 21 22 2 Abstract 23 The aim was to determine whether indirect exposure to pesticides, specifically a copper-based 24 fungicide, induces alterations in oxidative stress and subclinical and early kidney biomarkers in 25 male farmers tasked with olives harvesting. Furthermore, we tested whether sex influences the 26 susceptibility to pesticide-induced renal damage by comparing the results of this study with 27 those obtained previously. The study focused on olive farmers (n=41) indirectly exposed to 28 copper-based fungicides in Estepa (Sevilla, Spain), comparing them with a control group (n=32). 29 Blood samples were analyzed for metal concentrations (Cu, Mn, Se, and Zn), lipid peroxidation 30 (MDA), protein oxidation (carbonyl groups), and antioxidant enzyme activities (SOD and CAT) 31 while urine samples were assessed for biomarkers of early kidney damage (NGAL, KIM-1, 32 transferrin, IGFBP7, TIMP-2). Although no significant, a tendency to increase lipid and protein 33 oxidation was observed, together with the activity of antioxidant enzymes SOD and CAT, and a 34 decrease in total antioxidants. Moreover, an increase in urinary NGAL and IGFBP7 among 35 pesticide-exposed farmers suggests potential underdiagnosis of kidney damage. Farmers exhibit 36 a subtle tendency to oxidative stress compared to control, while metal levels are significantly 37 lower in farmers, suggesting potential compensatory responses. Furthermore, biomarkers for 38 early kidney damage are elevated, emphasizing their vulnerability in both sexes. These findings 39 highlight the need for investigations of renal health in pesticide-exposed farmers for 40 preventative measures and regular health monitoring. 41 42 43 Keywords: oxidative stress, metals, kidney, nephrotoxicity, farmers 44 45 3 1. Introduction 46 Agricultural workers are among the most vulnerable working populations due to social 47 and cultural risk factors such as ethnicity, immigration status, social class, and rural location 48 (Martín-Reina et al., 2021). In this sense, they meet different environmental conditions such as 49 exposure to heat and dehydration, pesticides, and heavy metals, and they are not always fully 50 protected. 51 Pesticides are widely used chemicals to preserve crops (Peres et al., 2006; Hassanin et 52 al., 2017). Exposure to them can contribute to both acute and chronic effects in humans, 53 including neurotoxicity, carcinogenicity, reproductive and endocrine disruption, neurological 54 damage, and immune system dysfunction, among others (Omland, 2002; Hassanin et al., 2017). 55 Concerning occupational exposure, the population is usually rural or greenhouse workers, and 56 manufacturing, mixing, or application workers (Groot and Van’t Hooft, 2016; Gangemi et al., 57 2016). On the other hand, the general population can be affected by environmental exposure, 58 consuming polluted food or drinking water, using pesticides at home, or living close to sprayed 59 fields (Gangemi et al., 2016). The type and severity of adverse health effects are determined by 60 the individual chemical category, the dose and the duration of exposure, the exposure route, and 61 the use of personal protective equipment (Martín-Reina et al., 2021). According to the World 62 Health Organization, long-term continued exposure causes around 772,000 new cases of 63 diseases per year (Hassanin et al., 2017). 64 Besides pesticides, rural workers can also be exposed to metals and metalloids. These 65 elements are characterized by their acute and chronic toxicity at extremely low doses and their 66 non-biodegradability but an exceptionally long biological half-life (Farcas et al., 2013; Quinteros 67 et al., 2013). In this sense, copper derivates such as copper sulfate have been commonly used in 68 agriculture for disease control such as “Repilo”, a fungal infection that causes significant 69 economic losses in the olive sector. In Spain, Repilo affects 60% of olive groves considered an 70 4 endemic disease in the provinces of Jaén, Córdoba, and Sevilla in the Andalusia region. Almost 71 all olive varieties cultivated in Andalusia are susceptible or highly susceptible (Junta de 72 Andalucía, 2010). Copper fungicides act as protectants by inhibiting conidia germination and 73 preventing infection establishment. Therefore, it is important to thoroughly spray the entire 74 canopy of the tree with the fungicide solution, especially the lower and inner branches where 75 the disease commonly develops. Their low phytotoxicity in olive trees and the necessity to keep 76 leaves protected for extended periods have led to regular annual treatments with relatively high 77 doses of copper. So, its long-term application leads to copper accumulation in olive soils, along 78 with remaining traces accumulated in leaves, posing a risk to worker health. Thus, it has been 79 demonstrated that copper-based fungicides persist on olive leaves even after being washed by 80 rain. This persistence varies among different types of fungicides (salts, hydroxides, oxychlorides, 81 oxides, and sulfates), with the most persistent being copper oxychloride (50-70%) and copper 82 sulfate-calcium sulfate (20%) (Marchal et al., 2003). 83 Copper (Cu) is an essential trace element that plays a physiological role in biological 84 systems, participating in electron transport and serving as a cofactor for numerous enzymes 85 involved in redox reactions (Jomova and Valko, 2011; Jomova et al., 2012; Dahiri et al., 2013). Cu 86 also protects against oxidative damage through ceruloplasmin and metallothionein (Sevcikova 87 et al., 2011; Dahiri et al., 2023). However, Cu can induce oxidative stress by generating reactive 88 oxygen species (ROS), leading to DNA damage and thiol-containing molecule oxidation like 89 glutathione. Prolonged indirect Cu exposure is associated with neurodegenerative and 90 cardiovascular diseases, hepatic disorders, and atherosclerosis, and some studies connect it with 91 nephrotoxicity (Pantic et al., 2019). 92 In addition, the general population exposure of the general population to metals and 93 metalloids can occur through the intake of contaminated drinking water or food; or through 94 lifestyle habits (smoking, alcohol consumption, drug abuse, or inhalation of contaminated air) 95 5 (Weyde et al., 2021; Dahiri et al., 2022). For instance, Zinc (Zn) acts as an antioxidant protecting 96 proteins from free radicals, reducing radical formation, inhibiting NADPH oxidase, and lowering 97 inflammatory cytokine production. Zn deficiency is linked to increased oxidative stress and 98 oxidation of lipids, proteins, and DNA (Jomova and Valko, 2011; Dahiri et al., 2023). Manganese 99 (Mn) in proteins contributes to metabolism, mitochondrial enzymes, and the antioxidant 100 superoxide dismutase (SOD). Excessive Mn exposure is associated with neurotoxicity (Bjørklund 101 et al., 2017; Dahiri et al., 2022). Selenium (Se) acts as an antioxidant cofactor for GPx, 102 selenoprotein P, and thioredoxin reductase, maintaining redox balance (Guo et al., 2018; Zakeri 103 et al., 2021; Dahiri et al., 2023). 104 As explained above, the mechanism of action for many pesticides and metals is the 105 generation of oxidative stress, as they can increase the production of free radicals that can 106 accumulate in the cell and damage biological macromolecules such as RNA, DNA, DNA repair 107 proteins among others, and affect antioxidant capacity and defense mechanisms, as well as 108 increase lipid peroxidation (Mostafalou and Abdollahi, 2013; Abdollahi et al., 2004; Possamai et 109 al., 2007; Mansour et al., 2009; Raina et al., 2009; Singh et al., 2011; Hernández et al., 2013; 110 Zepeda-Arce et al., 2017; Martín-Reina et al., 2021). At the organism level, these changes are 111 thought to be linked to long-term diseases such as carcinogenesis, neurodegeneration, and 112 cardiovascular, respiratory, renal, endocrine, and reproductive disorders (Jabłońska-Trypuć, 113 2017; Mostafalou and Abdollahi, 2013; Lozano-Paniagua et al., 2018). In addition, agrochemicals 114 such as pesticides and fertilizers, heavy metals, and occupational heat stress and dehydration 115 conditions have been postulated as risk factors for chronic kidney diseases (Chicas et al., 2019; 116 Valcke et al., 2019). In fact, there is an increasing prevalence of kidney diseases in farmers which 117 suggests a link between exposure to these compounds and impaired kidney function (Nagai, 118 2021). 119 6 It is required, thus, to study the repercussions associated with occupational exposure to 120 agrochemicals to formulate efficacious strategies for preventive measures against resultant 121 health issues. Most of the epidemiological studies about pesticide exposition are focused on 122 agricultural workers who handle and apply the pesticides on the field (direct exposure) but there 123 are hardly any studies about indirect exposition of workers collecting the products after the 124 waiting period recommended by pesticide manufacturers, who are chronically exposed 125 indirectly to pesticides trace levels (Martin-Reina et al., 2021). 126 In previous studies conducted in our laboratory subclinical kidney damage was observed 127 for the first time in a population of women in a rural setting with indirect chronic exposure to 128 pesticides (Martin-Reina et al., 2021). To delve further into these findings, the current study was 129 conducted to determine whether indirect exposure to pesticides, specifically a copper-based 130 fungicide, induces alterations in biochemical parameters. Thus, oxidative stress biomarkers, as 131 well as levels of metals, and subclinical and early kidney damage biomarkers were assessed in 132 male farmers tasked with olives harvesting. These results were then compared with a cohort of 133 men non-occupationally exposed to these chemicals but living in the same environment. A study 134 will also be conducted to examine the potential influence of sex on the risk of renal damage due 135 to pesticide exposure, comparing the results obtained in this study (males) with those of the 136 previously mentioned study (females). It is important to note that in both cases, the populations 137 are indirectly exposed to pesticides and are residents of the same rural area. 138 2. Materials and methods 139 2.1. Study population 140 The present study was focused on a cohort of farmers indirectly exposed to copper- 141 based fungicides through olive harvesting in the olive-producing regions of Estepa (n=41). These 142 fungicides are typically applied three times a year: at the beginning of autumn, the end of winter, 143 and in spring. The spring application is particularly critical for protecting new leaves. The main 144 7 trademark applied in this Area of Estepa is ZZ-cuprocol (70% copper oxychloride), and cuprosan 145 500 (50% copper oxychloride). Another cohort with similar characteristics, except for the 146 professional activity, was utilized as the control group which was classified as a non-occupational 147 exposed (NOE) group (n=32) 148 The olive-producing region of Estepa exhibits specific demographic features relevant to 149 this study. The population consists predominantly of individuals engaged in agricultural 150 activities, particularly olive cultivation. 151 Residents of the Estepa region typically maintain a stable residence for extended 152 periods, contributing to the homogeneity of the study sample. These demographic aspects 153 collectively contribute to the suitability of the Estepa region for investigating the potential health 154 impacts of indirect exposure to copper-based fungicides among olive farmers. 155 Recruitment took place during the harvesting season (September-October), coinciding 156 with health surveillance consultations. Regarding the control group, randomization was 157 determined by the participants' random entry into health surveillance medical consultations. 158 Inclusion criteria comprised males, farmers (and non-farmers for the control group), 159 aged 18-55 years, and residents in the reference area for at least the last 2 years. Workers with 160 chronic diseases such as diabetes, hypertension, or any type of renal or hepatic insufficiency 161 were excluded from the study, as these conditions could potentially alter the normal levels of 162 biomarkers considered in this study. The study was approved by the Research Ethics Committee 163 of the University of Sevilla in April 2022 (0984-N-22.) and written informed consent was obtained 164 from the participants. Once signed the informed consent, enrolled men, were asked to complete 165 a detailed questionnaire about demographics, lifestyles, dietary habits, and previous medical 166 history.2.2. Collection and processing of samples 167 8 Blood samples (≈ 20 mL) were collected in EDTA vacuum collection tubes (Vacutainer®) 168 and serum gel separation tubes (Vacutainer®) and placed in an icebox for transportation to the 169 laboratory. Upon arrival, the samples were promptly processed to prevent contamination and 170 hemolysis. 171 One of the EDTA tubes was employed to isolate the plasma using the method established 172 by the Spanish National Biobank Network (Martín-Reina et al., 2021). The obtained plasma was 173 transferred to multiple 1.5 mL tubes and stored at -80 ᵒC until measurements were conducted. 174 The gel separation tube was used to isolate serum following the proceduredescribed in 175 Dahiri et al. (2023). These serum samples were only used when plasma was unavailable for 176 measurements, and serum samples were allowed. 177 Whole blood samples were used for metal measurements. 178 Urine samples (≈125 mL) were transported under refrigeration (4 ᵒC) to the laboratory, 179 where they were subsampled and stored at - 80 ᵒC before analysis. 180 2.3. Determination of metals 181 The concentrations of Cu, Mn, Se, and Zn were evaluated in whole blood samples 182 collected in heparinized tubes. Blood digestion was carried out using the Anton Paar Multiwave 183 3000 system. Detection limits were determined by assessing the concentration of each element, 184 corresponding to a signal three times the standard deviation of a series of ten measurements of 185 the blank solution at the element peak. The average detection limits in blood were 0.51 ng/mL 186 for Cu, 0.21 ng/mL for Mn, 0.19 ng/mL for Se, and 5.46 ng/mL for Zn. 187 Prior to full digestion, samples underwent predigestion in the presence of HNO3 and 188 were subsequently incubated with added H2O2. The digestion program comprised an initial 189 power ramp from 0 to 800 W over 10 minutes, followed by a continuous power stage at 800 W 190 for 20 minutes, and a final cooling stage of 15 minutes, during which no power was applied. 191 15 308 A) B) C) 309 Figure 2. Catalase activity (A), SOD activity (B), and total antioxidant capacity found in NOE vs in farmers. 310 Data presented as mean ± SD.3.4. Evaluation of early kidney damage 311 Urinary excretion of subclinical and early kidney damage biomarkers is presented in 312 Figure 4. Overall, higher urinary excretions of all biomarkers were found in the Farmers group. 313 However, significantly higher levels were only identified for NGAL (p <0.05) and IGFBP7 (p <0.01). 314 315 0 5 10 15 20 25 NOE Farmers IU/g Cr NAG 0 20 40 60 80 100 120 140 NOE Farmers mg/g Cr Proteinuria * 0 5 10 15 20 25 30 35 40 NOE Farmers ng/mg Cr NGAL 0 1 2 NOE Farmers pg/mg Cr Transferrin ** 0 5 10 15 20 25 30 35 40 45 50 NOE Farmers ng/mg Cr IGFBP7 0 10 20 30 40 50 60 70 80 90 100 NOE Farmers pg2/mg2Cr IGFBP7 x TIMP-2 0 1 2 NOE Farmers pg/mg Cr KIM-1 0 1 2 3 NOE Farmers pg/mg Cr TIMP-2 16 Figure 4. Urinary biomarkers of renal damage. Data are presented as mean ± standard deviation. * p <0.05; 316 ** p <0.01 versus NOE group. Cr: creatinine; IGFBP7: insulin-like growth factor binding protein 7; KIM-1: 317 kidney injury molecule 1; NAG: N-acetyl-beta-D-glucosaminidase; NGAL: neutrophil gelatinase-associated 318 lipocalin; NOE: non-occupational exposed; TIMP-2: tissue inhibitor of metalloproteinases-2. 319 Despite observing evident early kidney damage in the Farmers group, the correlation 320 analysis shown in Table 3 reflected independence of this damage with the oxidative stress 321 biomarkers evaluated (only a significant correlation was observed between catalase activity and 322 NGAL (p<0.01) and between SOD and proteinuria and IGFBP7 (p<0.05 in both cases), although 323 with correlation coefficients indicating low linearity). 324 Table 3. Results of correlation analysis between biomarkers of oxidative stress (columns) and 325 those of early kidney damage (rows). Results are presented as Pearson's correlation coefficient (normally 326 distributed data) or Spearman's correlation coefficient (non-normally distributed data) (and their 327 associated p-value in parentheses). Significance of correlation: * p <0.05; ** p <0.01. IGFBP7: insulin-like 328 growth factor binding protein 7; KIM-1: kidney injury molecule 1; MDA: malondialdehyde; NAG: N-acetyl- 329 beta-D-glucosaminidase; NGAL: neutrophil gelatinase-associated lipocalin; SOD: superoxide dismutase; 330 TIMP-2: tissue inhibitor of metalloproteinases-2. 331 MDA Carbonyl groups Catalase activity SOD activity Total antioxidants Proteinuria 0.010 (0.942) 0.058 (0.681) 0.207 (0.136) 0.273 (0.046)* 0.080 (0.574) NAG -0.034 (0.806) 0.044 (0.754) 0.108 (0.443) 0.233 (0.090) -0.022 (0.879) NGAL 0.139 (0.316) 0.052 (0.712) 0.355 (0.009)** 0.179 (0.195) -0.219 (0.123) KIM-1 0.143 (0.303) 0.135 (0.335) 0.030 (0.830) -0.077 (0.581) -0.091 (0.525) Transferrin 0.194 (0.160) 0.198 (0.155) -0.14 (0.923) 0.137 (0.324) -0.185 (0.194) IGFBP7 0.160 (0.249) -0.117 (0.406) 0.177 (0.204) 0.305 (0.025)* -0.186 (0.192) TIMP-2 0.106 (0.444) 0.047 (0.736) -0.060 (0.672) -0.096 (0.492) -0.069 (0.630) IGFBP7 x TIMP-2 0.125 (0.369) -0.044 (0.754) 0.081 (0.564) 0.112 (0.422) -0.139 (0.329) In order to establish a possible influence of sex on the risk of kidney damage due to 332 pesticide exposure, a comparative analysis of the Farmers group with the group of female 333 17 farmers already evaluated in a previous study was performed (Martín-Reina et al., 2021). Those 334 biomarkers that had shown superior excretion in the Farmers group in both the current and 335 previous studies were compared (Figure 5). Different urinary excretion profiles were identified 336 between the male and female farmer groups. In fact, excretion of KIM-1 and transferrin was 337 significantly higher for males (p <0.01 and p <0.001, respectively), whereas it was significantly 338 higher for total protein and IGFBP7 for females (p <0.01). 339 340 Figure 5. Comparison of urinary excretion of some of the biomarkers of renal damage evaluated between 341 the male farmers group and female farmer group from our previous study. 1 Data are presented as box 342 plots. ** p <0.01; *** p <0.001 versus male farmers group. Cr: creatinine; IGFBP7: insulin-like growth factor 343 binding protein 7; KIM-1: kidney injury molecule 1; NAG: N-acetyl-beta-D-glucosaminidase; NGAL: 344 neutrophil gelatinase-associated lipocalin. 345 4. Discussion 346 ** *** ** ** 18 The present study determined potential indirect exposure to pesticides, specifically a 347 copper-based fungicide, and explored the possible association between occupational pesticide 348 exposure and alterations in biochemical parameters in two populations of rural men. 349 The investigated parameters included biomarkers of oxidative stress, levels of metals 350 associated with antioxidant function, Cu included, and early renal damage. Copper's significance 351 was heightened not only due to its physiological role but also because of its use as a fungicide in 352 olive trees. 353 The highest annual copper usage within the European countries has been reported in 354 Spain, with approximately 1,100 tons. The primary applications of this metal are in olives (51%), 355 almonds (23%), and grapes (20%), collectively representing 94% of copper use. Consequently, 356 Spain, along with Italy and France, presents the highest number of uses of this fungicide in 357 Europe (Tamm et al., 2022). 358 The study was conducted in the Estepa area (Seville, Spain), where 70% of the land is 359 covered by olive trees, generating 225 million Euros annually and setting a standard for social 360 and economic development in line with national and European standards (Sousa et al., 2020; 361 Land 2020). This study can be considered the first one focusing on the health of olive crop sector 362 farmers who are indirectly exposed to trace levels of copper. 363 Our research contributes significant data on the possible relationship between indirect 364 pesticide exposure and health effects in a farming population compared with a NOE group. 365 Previous studies in a female farmer population from this region revealed that chronic indirect 366 pesticide exposure led to subclinical tubular damage in a rural setting, potentially progressing to 367 chronic kidney disease. Additionally, oxidative stress biomarkers measured were higher in the 368 female farmer group compared to the NOE group (Martin-Reina et al., 2021). Demographic data 369 indicated that approximately 67% of our workers had been indirectly exposed to pesticides for 370 more than 10 years. Most of them reported using personal protective equipment such as gloves 371 and masks, while none reported the use of glasses. Similarly, most of the female farmers from 372 19 this area reported the use of gloves; however, none of them reported the use of masks and 373 glasses (Martin-Reina et al., 2021). 374 Concerning the studies of the influence of metals on oxidative stress in workers exposed 375 to pesticides, the number of publications is scarce. In the present study, not only were there no 376 higher blood levels in workers exposed to traces that might remain on copper sulfate-treated 377 leaves, but significantly higher levels were observed in the control group population. Similar 378 trends were observed for other studied metals (Mn, Se, and Zn), all recognized for their 379 antioxidant function. The detection of these levels of metals could compensate for the potential 380 oxidative stress induced by the chronic indirect exposure of these farmers to pesticides, being a 381 possible reason for the lack of statistically significant results on the antioxidant parameters. 382 Regarding this fact, some authors suggested the efficacy of Zn and Se to inhibit the Cd-dependent 383 intracellular signaling pathway leading to oxidative stress in SH-SY5Y cells (Branca et al., 2018). 384 On the contrary, other authors observed that higher levels of Cu, Zn, and their mixture decreased 385 significantly the activities of CAT and SOD in G. mellonella larvae which are used as an alternative 386 organism to vertebrates in studies of human acute toxicity (Mese et al., 2022). 387 The use of pesticides leads to the generation of ROS which have harmful effects on 388 human health. The higher concentration and prolonged exposure to these oxidants induced by 389 pesticides lead to oxidative damage of cellular macromolecules, including lipids, DNA, and 390 proteins, and/or cellular death. This mechanism of pesticide toxicity has become a focal point in 391 toxicological research, especially when ROS cannot be removed by antioxidant defense systems 392 (Oju et al., 2011; Zepeda-Arce et al., 2017; Nascimento et al., 2021). For instance, Zepeda-Arce 393 et al. (2017) measured SOD, CAT, GPx, and GR levels in rural workers, and genotoxicity by the 394 comet assay, obtaining a decrease of SOD and CAT in workers compared to the controls, and a 395 correlation between MDA and GPx. Furthermore, this study also reported significant differences 396 between long-term and temporary workers, and a positive correlation between DNA damage 397 and MDA levels in those exposed for a long term (Zepeda-Arce et al., 2017). 398 20 Most studies on the toxic effects of pesticides focus on applicators; however, exposure 399 to these toxicants occurs not only during application but also during handling, product 400 preparation, and harvesting (Lovinson et al., 2021; Martin-Reina et al., 2021). There are no 401 previous studies in the literature on oxidative stress in farmers collecting olives and exposed to 402 copper-based fungicides. Increased levels of oxidative stress have been demonstrated in various 403 farmer populations globally. For instance, farmers exposed to pesticides in Jalisco, Mexico, 404 reported elevated LPO levels and carbonyl groups compared to the unexposed group. These 405 results provide a theoretical foundation for explaining the high prevalence of neurodegenerative 406 diseases in this region of Mexico (Salazar-Flores et al., 2020). Similar findings were reported by 407 Lovison-Sasso et al. (2021) in a rural region of Brazil, where a significant increase in LPO and 408 carbonylated protein formation was observed in the exposed group. However, unlike our results, 409 the levels of CAT and SOD were significantly decreased compared to the control group, 410 suggesting an unsuccessful antibody stimulation of the antioxidant defense system. Additionally, 411 in a Brazilian rural population, levels of LPO and carbonyl showed lower values in the pesticide- 412 using group, along with a significant reduction in SOD activity, indicating a physiological response 413 to chronic pesticide exposure (Cattelan et al., 2018). 414 The kidney is especially vulnerable to damage caused by pesticides since it is one of the 415 main organs involved in their elimination (Green et al., 2004). Despite this, there are no 416 conclusive results, since some studies include populations with kidney damage risk factors such 417 as heat stress and dehydration. It is also difficult to establish this association when pesticide 418 exposure differs in type, number, and time. 419 On the other hand, kidney damage could be underdiagnosed in these studies since the 420 diagnosis is based on the increase in creatinine, and kidney dysfunction is only evident when it 421 has decreased by 50% (Bagshaw and Bellomo, 2007). Therefore, in this work, possible subclinical 422 kidney damage was evaluated using a panel of biomarkers of early kidney damage. These 423 21 biomarkers have been used to localize specific segments of injured tubules and have diverse 424 roles in pathophysiological processes in acute kidney injury, including inflammation, repair, and 425 fibrosis. Current evidence suggests that these biomarkers could even be used to predict the 426 transition to chronic kidney disease (CKD) (Yu and Bonventre, 2020). 427 Our results showed an increase in NGAL and IGFBP7 in pesticide-exposed farmers, 428 without alteration of creatinine levels (data not shown). In this regard, NGAL is a small 429 polypeptide resistant to proteases and can therefore be easily detected in urine. NGAL, under 430 physiological conditions, is filtered at the glomerular level and reabsorbed in the proximal 431 tubules, so an increase in its excretion in the urine may be due to an increase in its de novo 432 synthesis in the distal tubular epithelium in response to damage, or a failure in the reabsorption 433 process as a consequence of proximal tubular damage. Consequently, increased levels of this 434 protein in urine are indicative of proximal and/or distal tubular kidney damage (Tonomura et al., 435 2014). Regarding IGFBP7, it is expressed in tubular cells in response to DNA damage and other 436 forms of injury (Kellum and Chawla, 2015). Due to its molecular size of 30 kDa, IGFBP-7 may be 437 freely filtered. Reduced tubular reabsorption and/or increased secretion of IGFBP-7, or its 438 release from damaged tubular epithelium, might be the cause of higher urinary excretion in our 439 study (farmers). Therefore, the pathophysiological significance of the excretion of both 440 biomarkers suggests that exposure to these pesticides can lead to subclinical tubular injury. This 441 finding supports the results found in a previous study from our laboratory in which we found in 442 female farmers exposed to pesticides an increase in biomarkers that suggested subclinical 443 tubular damage (Martín-Reina et al., 2021). Moreover, another study carried out on greenhouse 444 workers obtained results like ours (Lozano-Paniagua et al., 2023). The importance of it is the risk 445 that this damage poses for progressing to CKD. Therefore, it would be interesting to validate 446 these two biomarkers in future studies to confirm their usefulness as diagnostic tools in the 447 periodic medical check-ups of these farmers and take preventive measures against kidney 448 damage. 449 22 On the other hand, the results related to the lack of correlation between the biomarkers 450 of oxidative stress and those of early kidney damage could suggest that other mechanisms are 451 involved in the nephrotoxicity associated with copper (the main component of the fungicides 452 studied) (Zou et al., 2024). 453 Regarding the possible influence of sex on the risk of kidney damage due to pesticide 454 exposure, our results identified different urinary excretion profiles of biomarkers between male 455 and female farmer groups. Despite this, the pathophysiological significance is similar since in 456 both cases there is excretion of biomarkers of tubular damage and the excretion of proteins in 457 women reflects non-specific damage that could have the same origin. However, these results 458 open the possibility of approaching these studies from a gender perspective to delve deeper into 459 this aspect. 460 Conclusion 461 This study explores the impact of indirect pesticide exposure, focusing on a copper-based 462 fungicide, on rural men involved in olive harvesting. Strikingly, metal levels, especially copper, 463 are significantly lower in farmers, challenging expectations and suggesting potential 464 compensatory responses. Notably, biomarkers for early kidney damage are elevated, which 465 suggests subclinical tubular damage and emphasizes the vulnerability of the kidneys to pesticide 466 exposure in both men and women. These findings highlight the need for targeted investigations 467 into renal health in pesticide-exposed farmers, advocating for preventative measures and regular 468 health monitoring. 469 Acknowledgments: The authors acknowledge the disinterested participation of the 470 men under study. 471 Funding sources: This research was funded by Prevent Foundation, grant number 472 4465/0828 and by the Andalusian Institute of Occupational Risk Prevention (CTC-2022140473) 473 23 Ethical approval 474 Ethical clearance was obtained from the Committee of Ethics of Researching of the 475 University of Seville (code: 0984-N-22.June 2022) and agreed with the Declaration of Helsinki for 476 International Health Research. 477 Consent to participate 478 Written informed consent was also obtained from all the participants after being 479 informed about the objectives of the study and the right to drop out of the study at any time. 480 Consent to publish 481 Non-applicable. 482 Competing interests 483 None 484 Authors contributions 485 Study conception and design were performed by Isabel M. Moreno, Juan D. 486 Bautista, Ana I. Morales. Collection of the samples was carried out by José Martín-Reina, 487 Raúl Aguilera-Velázquez. Material preparation, data collection and analysis were 488 performed by Alfredo G. Casanova, María G. Hinojosa, Carmen Chamorro-López. The 489 first draft of the manuscript was written by María G. Hinojosa and Isabel M. Navarro, and 490 all authors commented on previous versions of the manuscript. All authors read and 491 approved the final version of the manuscript. 492 493 Bibliography 494 24 Adedeji, A.O.; Pourmohamad, T.; Chen, Y.; Burkey, J.; Betts, C.J.; Bickerton, S.J.; Sonee, M.; 495 McDuffie, J.E. Investigating the Value of Urine Volume, Creatinine, and Cystatin C for Urinary 496 Biomarkers Normalization for Drug Development Studies. Int J Toxicol. 2019;38:12–22. Doi: 497 10.1177/1091581818819791. 498 Bagshaw, S.M.; Bellomo, R. Early diagnosis of acute kidney injury. Curr. Opin. Crit. Care 499 2007;13:638–644. Doi: 10.1097/MCC.0b013e3282f07570. 500 Bjørklund, G.; Chartrand, M.S.; Aaseth, J. Manganese exposure and neurotoxic effects in 501 children. Environ. Res. 2017;155:380–384. Doi: 10.1016/j.envres.2017.03.003. 502 Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of 503 protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72: 248–254. Doi: 504 10.1006/abio.1976.9999. 505 Branca, J.J.V.; Morucci, G.; Maresca, M.; Tenci, B.; Cascella, R.; Paternostro, F.; Ghelardini, C.; 506 Gulisano, M.; Manelli, L.D.C.; Pacini, A. Selenium and zinc: two key players against cadmium- 507 induced neuronal toxicity. Toxicol in Vitro. 2018;48:159-169. Doi: 10.1016/j.tiv.2018.01.007. 508 Cattelan, M.D.P.; Maurer, P.; Garcia, F.; Berro, L.F.; Machado, M.M.; Manfredini, V.; Piccoli, J.D.C.E. 509 Occupational exposure to pesticides in family agriculture and the oxidative, biochemical and 510 hematological profile in this agricultural model. Life Sciences. 2018;203:177-183. Doi: 511 10.1016/j.lfs.2018.04.038. 512 Chicas, R.; Mix, J.; Mac, V.; Flocks, J.; Dickman, N.E.; Hertzberg, V.; McCauley, L. Chronic kidney 513 disease among workers. Bjørklund, G.; Chartrand, M.S.; Aaseth, J. Manganese exposure and 514 neurotoxic effects in children. Workplace Health Saf. 2019;67:481-490. Doi: 515 10.1177/2165079919843308. 516 Dahiri, B.; Hinojosa, M.G.; Carbonero-Aguilar, P.; Cerrillos, L.; Ostos, R.; Bautista, J.; Moreno, I. 517 Assessment of the oxidative status in mother-child couples from Seville (Spain): A prospective 518