Avaliação de risco agudo à exposição dietética a resíduos de agrotóxicos em tomate / Acute risk assessment of dietary exposure to pesticide residues in tomato

A utilização de agrotóxicos ao redor do mundo é elevada, estimada em mais 2 milhões de toneladas e somente o continente americano emprega 1,2 milhão de toneladas de produtos. O Brasil possui na agricultura a sua principal atividade econômica e utilizou em 2021 aproximadamente 700 mil toneladas de agrotóxicos. O tomate é um dos vegetais mais cultivados e consumidos no mundo e o Brasil figura como o 10º maior produtor. O consumo anual médio de tomate do brasileiro é de 4,2 kg, é um vegetal nutritivo e com propriedades associadas à prevenção de câncer. O tomate é uma das culturas com maior uso de agrotóxicos e durante o período desse estudo, era autorizado o uso de 123 agrotóxicos. Os resíduos desses produtos nos alimentos podem acarretar em diversos problemas à saúde, mesmo em curta exposição (< 24h). Desse modo, uma das maneiras de garantir a segurança alimentar da população é a realização da avaliação de risco de contaminação dietética. No processo de validação 73 ingredientes ativos respeitaram os critérios do protocolo adotado. Para a realização da estimativa de risco de exposição dietética aguda, foram coletadas 30 amostras de tomate in natura e 11 de tomate pelado, entre setembro de 2021 e março de 2022. Para a extração dos compostos de interesse foi utilizado o método QuEChERS e para avaliação dos resíduos, a cromatografia líquida acoplada à espectrometria de massas. Das amostras de tomate in natura, somente seis (20 %) estavam isentas dos compostos pesquisados; 18 (60 %) apresentaram resíduo(s) abaixo do limite (LMR) estabelecido e seis amostras (20 %) foram consideradas impróprias ao consumo. Das amostras de tomate pelado, três estavam isentas dos agrotóxicos pesquisados e oito (72 %) apresentaram resíduo de carbendazim abaixo do LMR. Nenhuma das amostras mostrou potencial de contaminação aguda por agrotóxicos, apesar disso não é possível afirmar que não há risco, pois não há como estimar os potenciais efeitos adversos provenientes do consumo de um alimento com múltiplos compostos.

More than 2 million tons of pesticide products are used annually through the world and only the America continent was used 1,2 million tons of these products. The agriculture is the main economic activity in Brazil, this way in 2021 approximately 700 thousand tons of pesticide were applied in its crops. Tomato is one of the most cultivated and consumed vegetables in the world and Brazil is the 10th largest producer. The Brazilian people consumes an average of 4.2 kg of tomatoes by year, it is a nutritious vegetable with anti-cancer properties. Tomato crop is one of the which highest pesticide usages, during this study 123 compounds were permitted for this crop. Pesticide residues in food may causes several health problems, even in short-term exposition (< 24h), thus one of the ways to ensure the food safety to population is performing the dietary contamination risk assessment. In the validation process 73 active ingredients were within the established criteria. To perform the acute risk assessment of dietary exposure, were collected samples of: fresh tomato (30) and whole peeled tomato (11) in between September of 2021 and March of 2022. To extraction of interest substances was used the QuEChERS method and to residue evaluation the liquid chromatography coupled to mass spectrometry. Of the fresh tomato samples, only six (20 %) were free of searched analytes; 18 (60 %) showed residue(s) bellow the established limits (MRL) and six (20 %) were considered unfit to consumption. Of the whole peeled tomato samples, three were free of searched substances and eight (72 %) showed residue of carbendazim bellow the MRL. None of the samples showed potential for acute contamination by pesticide, however it is not possible to say that there is no risk, as there is no way to estimate the potential adverse effects on health arising from the consumption of a food with several compounds.

Towards achieving a modern science-based paradigm for agrochemical carcinogenicity assessment.

The rodent cancer bioassay has been the standard approach to fulfill regulatory requirements for assessing human carcinogenic potential of agrochemicals, food additives, industrial chemicals, and pharmaceuticals. Decades of research have described the limitations of the rodent cancer bioassay leading to international initiatives to seek alternatives and establish approaches that modernize carcinogenicity assessment. Biologically relevant approaches can provide mechanistic information and increased efficiency for evaluating hazard and risk of chemical carcinogenicity to humans. The application of human-relevant mechanistic understanding to support new approaches to carcinogenicity assessment will be invaluable for regulatory decision-making. The present work outlines the challenges and opportunities that authorities should consider as they come together to build a roadmap that leads to global acceptance and incorporation of fit-for-purpose, scientifically defensible new approaches for human-relevant carcinogenicity assessment of agrochemicals.

Comercialização de agrotóxicos e desfechos de saúde no Estado do Paraná: uma associação não linear / Marketing of Pesticides and Health Outcomes in the State of Paraná: A Nonlinear Association

Resumo Este estudo ecológico teve por objetivo analisar a associação entre a quantidade de comercialização de agrotóxicos e alguns agravos e causas de mortalidade no estado do Paraná no período de 2013 a 2017. Desta forma, realizou-se um comparativo entre as 22 regiões de saúde, identificando a relação entre a taxa de comercialização de agrotóxicos por habitante com as seguintes variáveis: taxa de intoxicações exógenas relacionadas ao trabalho, taxa de intoxicações exógenas por agrotóxicos relacionadas ao trabalho, taxa de tentativas de suicídio e taxas de mortalidade por neoplasias, malformação congênita e suicídio. Para estes três últimos desfechos de saúde, os modelos não lineares selecionados tiveram seus R-quadrado acima de 0,500. A Regional de Saúde de Cascavel se destacou com mais municípios com altas taxas de comercialização de agrotóxicos e desfechos de saúde, ao contrário da Regional de saúde Metropolitana. Observa-se que o perfil dos municípios que têm maiores taxas dos agravos estudados é rural e com produção majoritariamente de soja, milho, trigo, fumo, pastagens e feijão. Dessa forma, ressalta-se a necessidade de rever o modelo agroalimentar convencional e promover políticas públicas rigorosas para controle dos agrotóxicos, além de prevenção de agravos à saúde, fortalecendo os serviços de Vigilância Epidemiológica.

Abstract This ecological study aimed to analyze the association between the amount of pesticide commercialization and some diseases and causes of mortality in the state of Paraná from 2013 to 2017. In this way, a comparison was made between the 22 health regions, identifying the relationship between the rate of commercialization of pesticides per inhabitant with the following variables: rate of exogenous intoxication related to work, rate of exogenous intoxication by pesticides related to work, rate of suicide attempts and mortality rates from neoplasms, congenital malformation and suicide. For these last three health outcomes, the selected nonlinear models had their R-squared above 0.500. The Cascavel Health Region stood out with more municipalities with high rates of pesticide marketing and health outcomes, unlike the Metropolitan Health Region. It is observed that the profile of the municipalities that have the highest rates of the diseases studied is rural and with production mainly of soy, corn, wheat, tobacco, pastures, and beans. Thus, the need to review the conventional agri-food model and promote rigorous public policies to control pesticides, in addition to preventing health problems, strengthening Epidemiological Surveillance services is highlighted.

Chronic consumption of contaminated feed with 2 4 dichlorophenoxyacetic acid (2 4 D) herbicide in rodents: effects on male reproductive health / Consumo crônico de ração contaminada com herbicida ácido 2 4 di clorofenoxiacético (2 4 D) em roedores: efeitos na saúde reprodutiva masculina

Introduction: The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is one of the most widely used pesticides in the world. There is evidence that this herbicide can induce deleterious effects in non-target organisms, including impairment of reproduction function. Objective: The aim of this study was to evaluate the reproductive effects of the chronic consumption of contaminated feed with 2,4-D in rats using food environmental spraying simulation. Methods: Animals orally exposed received nebulized chow with 2,4-D solution in different concentrations for 180 days: 0 (control - CG), 20.69 (LCG), 34.63 (MCG), or 51.66 ppm day−1 (HCG). Results: Sperm quality was impaired to 2,4-D. The percentage of sperm with progressive movement, number of sperm in the testis and daily sperm production were decreased in all exposed groups to the herbicide compared to CG. Sperm counts in the caput/corpus and cauda epididymis were reduced in MCG and HCG, and sperm transit time was delayed in the epididymis of LCG. There was a negative impact on sperm morphology and plasma membrane integrity in MCG and HCG, respectively. Germ cell exfoliation within the lumen of the seminiferous tubules and epithelial vacuolization in epididymis were found in the HCG. Conclusion: This is the first study to describe the negative impact on male reproductive morphophysiology after chronic exposure to 2,4-D using food nebulization in environmentally relevant concentrations, based on agronomic use of the herbicide. The reproductive injuries identified raise concerns about the impacts of wide population exposure to 2,4-D (AU).

Introdução: O herbicida 2,4- ácido diclorofenoxiacético (2,4-D) é um dos agrotóxicos mais utilizados no mundo. Há evidências de que este herbicida pode induzir efeitos deletérios em organismos não-alvo, incluindo prejuízo na função reprodutiva. Objetivo: O objetivo deste estudo foi avaliar os efeitos reprodutivos do consumo crônico de ração contaminada com 2,4-D em ratos, utilizando simulação de pulverização ambiental de alimentos. Métodos: Animais expostos oralmente receberam ração nebulizada com solução de 2,4-D em diferentes concentrações por 180 dias: 0 (controle - GC), 20,69 (LCG), 34,63 (MCG) ou 51,66 ppm dia−1 (HCG). Resultados: A qualidade espermática foi prejudicada pelo 2,4-D. A porcentagem de espermatozoides com movimento progressivo, número de espermatozoides no testículo e produção diária de espermatozoides foram menores em todos os grupos expostos ao herbicida, quando comparados ao GC. A contagem de espermatozoides na cabeça/corpo e cauda do epidídimo foi reduzida em MCG e HCG, e o tempo de trânsito espermático atrasou no epidídimo em LCG. Houve impacto negativo na morfologia espermática e na integridade da membrana plasmática em MCG e HCG, respectivamente. Esfoliação de células germinativas no lúmen dos túbulos seminíferos e vacuolização epitelial no epidídimo foram encontradas em HCG. Conclusão: Este é o primeiro estudo a descrever o impacto negativo na morfofisiologia reprodutiva masculina após exposição crônica ao 2,4-D, utilizando nebulização de alimentos em concentrações ambientalmente relevantes, com base no uso agronômico do herbicida. As lesões reprodutivas identificadas levantam preocupações sobre os impactos da ampla exposição da população ao 2,4-D (AU).

Combined toxicities of cadmium and five agrochemicals to the larval zebrafish (Danio rerio).

Different pollutants usually co-exist in the natural environment, and the ecological and health risk assessment of agrochemicals needs to be carried out based on the combined toxicological effects of pollutants. To examine the combined toxicity to aquatic organisms, the effects of cadmium (Cd) and five pesticides (acetamiprid, carbendazim, azoxystrobin, chlorpyrifos, and bifenthrin) mixture on zebrafish (Danio rerio) larvae were assessed. The data from the 96-h toxicity test indicated that bifenthrin possessed the highest toxicity to D. rerio with the LC50 value of 0.15 mg L-1, followed by chlorpyrifos (0.36 mg L-1) and azoxystrobin (0.63 mg L-1). Cd (6.84 mg L-1) and carbendazim (8.53 mg L-1) induced the intermediate toxic responses, while acetamiprid (58.39 mg L-1) presented the lowest toxicity to the organisms. Pesticide mixtures containing chlorpyrifos and bifenthrin or acetamiprid and carbendazim showed synergistic impacts on the zebrafish. Besides, two binary combinations of Cd-acetamiprid and Cd-chlorpyrifos also displayed a synergistic effect on D. rerio. Our results offered a better idea of the mixed ecological risk assessment of Cd and different agricultural chemicals to aquatic organisms. Our findings better interpreted how the interaction between Cd and various agrochemicals changed their toxicity to aquatic vertebrates and provided valuable insights into critical impacts on the ecological hazard of their combinations.

Rethinking chronic toxicity and carcinogenicity assessment for agrochemicals project (ReCAAP): A reporting framework to support a weight of evidence safety assessment without long-term rodent bioassays.

Rodent cancer bioassays have been long-required studies for regulatory assessment of human cancer hazard and risk. These studies use hundreds of animals, are resource intensive, and certain aspects of these studies have limited human relevance. The past 10 years have seen an exponential growth of new technologies with the potential to effectively evaluate human cancer hazard and risk while reducing, refining, or replacing animal use. To streamline and facilitate uptake of new technologies, a workgroup comprised of scientists from government, academia, non-governmental organizations, and industry stakeholders developed a framework for waiver rationales of rodent cancer bioassays for consideration in agrochemical safety assessment. The workgroup used an iterative approach, incorporating regulatory agency feedback, and identifying critical information to be considered in a risk assessment-based weight of evidence determination of the need for rodent cancer bioassays. The reporting framework described herein was developed to support a chronic toxicity and carcinogenicity study waiver rationale, which includes information on use pattern(s), exposure scenario(s), pesticidal mode-of-action, physicochemical properties, metabolism, toxicokinetics, toxicological data including mechanistic data, and chemical read-across from similar registered pesticides. The framework could also be applied to endpoints other than chronic toxicity and carcinogenicity, and for chemicals other than agrochemicals.

Conflictividad socio-ambiental por uso de agroquímicos en Salta, Santiago del Estero y Santa Fe, Argentina / Social and environmental conflicts caused by agrochemical use in Salta, Santiago del Estero and Santa Fe, Argentina

Resumen El objetivo del artículo es contribuir a la comprensión y visibilización de los conflictos y controversias en torno al uso de agroquímicos en las provincias de Santa Fe, Santiago del Estero y Salta, Argentina. Se relevaron y sistematizaron fuentes de información secundaria, con el fin de llevar adelante un primer análisis contextual de los marcos regulatorios, las políticas públicas y la emergencia de procesos de construcción social de los riesgos ambientales y sanitarios. El análisis fue organizado en torno a tres dimensiones: normativa, político-institucional y territorial-sanitaria. En las tres jurisdicciones existen leyes específicas que regulan el uso de agroquímicos y cierta institucionalidad destinada a su implementación, control y monitoreo. No obstante, y en concordancia con lo ocurrido a nivel regional e internacional, el estudio relevó una multiplicidad de situaciones y/o eventos conflictivos que problematizan los impactos ambientales y sanitarios. La política relativa a la utilización de agroquímicos está fragmentada en múltiples regulaciones, instituciones y escalas de competencias, marco dentro del cual la política sanitaria y ambiental poseen un lugar rezagado. Más allá de ciertos avances, no hay reconocimiento oficial del daño a la salud y el ambiente generado por el uso de agroquímicos.

Abstract The purpose of this article is to contribute to the understanding and visibility of conflicts and disputes over the use of agrochemicals in the provinces of Santa Fe, Santiago del Estero and Salta, in Argentina. Secondary information sources were gathered and systematized to perform a first contextual analysis of regulatory frameworks, public policies and the emergence of social construction processes related to environmental and health risks. This analysis revolved around three dimensions: a regulatory dimension, a political-institutional dimension, and a territorial-health dimension. In all three jurisdictions, there are specific laws that govern the use of agrochemicals and certain institutionalization intended to implement, control and monitor them. However, similarly to what has happened at the regional and international levels, the study revealed multiple conflict situations and/or events that call environmental and health impacts into question. Agrochemical use policy is shattered into multiple regulations, institutions and levels of competence, a framework in which health and environmental policies are left behind. Despite some progress, there is no official recognition of the health and environmental damage caused by the use of agrochemicals.

Rethinking agrochemical safety assessment: A perspective.

Agrochemical safety assessment has traditionally relied on the use of animals for toxicity testing, based on scientific understanding and test guidelines developed in the 1980s. However, since then, there have been significant advances in the toxicological sciences that have improved our understanding of mechanisms underpinning adverse human health effects. The time is ripe to 'rethink' approaches used for human safety assessments of agrochemicals to ensure they reflect current scientific understanding and increasingly embrace new opportunities to improve human relevance and predictivity, and to reduce the reliance on animals. Although the ultimate aim is to enable a paradigm shift and an overhaul of global regulatory data requirements, there is much that can be done now to ensure new opportunities and approaches are adopted and implemented within the current regulatory frameworks. This commentary reviews current initiatives and emerging opportunities to embrace new approaches to improve agrochemical safety assessment for humans, and considers various endpoints and initiatives (including acute toxicity, repeat dose toxicity studies, carcinogenicity, developmental and reproductive toxicity, exposure-driven approaches, inhalation toxicity, and data modelling). Realistic aspirations to improve safety assessment, incorporate new technologies and reduce reliance on animal testing without compromising protection goals are discussed.

An evaluation of carcinogenicity predictors from short-term and sub chronic repeat-dose studies of agrochemicals in rats: Opportunities to refine and reduce animal use.

The aim of this study was to examine whether short term, repeat dose, rat studies provide sufficient information about potential carcinogenicity to enable predictions about the carcinogenic potential of agrochemicals to be made earlier in compound development. This study aimed to identify any correlations between toxicity findings obtained for short term rat studies (28 day and 90 day) and neoplastic findings obtained from 24 month rat carcinogenicity studies for agrochemical compounds (18 compounds) tested in Han Wistar and Sprague Dawley rats. The macroscopic pathology, microscopic pathology, hematology, biochemistry, organ weights, estrogen receptor activation and genotoxicity results were examined. Seven out of 18 non genotoxic compounds developed tumors in treated rats in the carcinogenicity study and of these, two compounds showed no preneoplastic findings in the affected tissues (false negatives). Of the remaining five true positives, correlations were noted between corneal opacity and keratitis (90 day study) as early indicators of squamous cell carcinoma and papilloma of the cornea of the eye (compound 1, a hydroxyphenylpyruvate dioxygenase inhibitor) and inflammation of the stomach and kidney (90 day study) and gastric squamous cell papilloma and squamous cell carcinoma and renal tubular adenoma and carcinoma, respectively (compound 12, a fungicide with multisite activity). Minor decreases in uterine weight and increases in estradiol hydroxylation activity at 28 days were associated with endometrial adenocarcinoma (compound 18, a mitochondrial complex II electron transport inhibitor). Early liver weight increases and hepatocellular centrilobular hypertrophy (28 day study) were associated with thyroid follicular adenomas (compound 11, a succinate dehydrogenase inhibitor) in female animals only. Hepatic centrilobular hypertrophy (28 day studies) correlated with thyroid adenomas in males in carcinogenicity studies (compound 2, a hydroxyphenylpyruvate dioxygenase inhibitor). In contrast, treatment related, nasopharynx tumors (compound 3, an elongase inhibitor) and uterine adenocarcinoma (compound 9, a succinate dehydrogenase inhibitor) could not be correlated with findings from the short term studies examined. Eleven compounds displayed preneoplastic findings with no tumors (false positives) and there were no compounds with no preneoplastic findings and no tumors (true negatives). This work indicates the value of examining historical, short term studies for specific, nonneoplastic findings which correlate with tumors in carcinogenicity studies, which may obviate the need for further animal carcinogenicity studies.

Comparison of the predictive nature of the Genomic Allergen Rapid Detection (GARD) assay with mammalian assays in determining the skin sensitisation potential of agrochemical active ingredients.

Alternatives to mammalian testing are highly desirable to predict the skin sensitisation potential of agrochemical active ingredients (AI). The GARD assay, a stimulated, dendritic cell-like, cell line measuring genomic signatures, was evaluated using twelve AIs (seven sensitisers and five non-sensitisers) and the results compared with historical results from guinea pig or local lymph node assay (LLNA) studies. Initial GARD results suggested 11/12 AIs were sensitisers and six concurred with mammalian data. Conformal predictions changed one AI to a non-sensitiser. An AI identified as non-sensitising in the GARD assay was considered a potent sensitiser in the LLNA. In total 7/12 GARD results corresponded with mammalian data. AI chemistries might not be comparable to the GARD training set in terms of applicability domains. Whilst the GARD assay can replace mammalian tests for skin sensitisation evaluation for compounds including cosmetic ingredients, further work in agrochemical chemistries is needed for this assay to be a viable replacement to animal testing. The work conducted here is, however, considered exploratory research and the methodology needs further development to be validated for agrochemicals. Mammalian and other alternative assays for regulatory safety assessments of AIs must provide confidence to assign the appropriate classification for human health protection.

A rat subchronic study transcriptional point of departure estimates a carcinogenicity study apical point of departure.

Considerations of human relevance and animal use are driving research to identify new approaches to inform risk assessment of chemicals and replace guideline-based rodent carcinogenicity tests. Here, the hypothesis was tested across four agrochemicals that 1) a rat 90-day transcriptome-based BEPOD is protective of a rat carcinogenicity study and 2) a subchronic liver or kidney BEPOD would approximate a cancer bioassay apical POD derived from other organs and a rat subchronic BEPOD would approximate a mouse cancer bioassay apical POD. Using RNA sequencing and BMDExpress software, liver and/or kidney BEPOD values were generated in male rats exposed for 90 days to either Triclopyr Acid, Pronamide, Sulfoxaflor, or Fenpicoxamid. BEPOD values were compared to benchmark dose-derived apical POD values generated from rat 90-day and rodent carcinogenicity studies. Across all four agrochemicals, findings showed that a rat 90-day study BEPOD approximated the most sensitive apical POD (within 10-fold) generated from the 90-day rat study and long-term rodent carcinogenicity studies. This study supports the conclusion that a subchronic transcriptome-based BEPOD could be utilized to estimate an apical POD within a risk-based approach of chronic toxicity and carcinogenicity agrochemical assessment, abrogating the need for time- and resource-intensive rodent carcinogenicity studies and minimizing animal testing.

Identification of early liver toxicity gene biomarkers using comparative supervised machine learning.

Screening agrochemicals and pharmaceuticals for potential liver toxicity is required for regulatory approval and is an expensive and time-consuming process. The identification and utilization of early exposure gene signatures and robust predictive models in regulatory toxicity testing has the potential to reduce time and costs substantially. In this study, comparative supervised machine learning approaches were applied to the rat liver TG-GATEs dataset to develop feature selection and predictive testing. We identified ten gene biomarkers using three different feature selection methods that predicted liver necrosis with high specificity and selectivity in an independent validation dataset from the Microarray Quality Control (MAQC)-II study. Nine of the ten genes that were selected with the supervised methods are involved in metabolism and detoxification (Car3, Crat, Cyp39a1, Dcd, Lbp, Scly, Slc23a1, and Tkfc) and transcriptional regulation (Ablim3). Several of these genes are also implicated in liver carcinogenesis, including Crat, Car3 and Slc23a1. Our biomarker gene signature provides high statistical accuracy and a manageable number of genes to study as indicators to potentially accelerate toxicity testing based on their ability to induce liver necrosis and, eventually, liver cancer.

Towards a mechanism-based approach for the prediction of nongenotoxic carcinogenic potential of agrochemicals.

Chemical substances are subjected to assessment of genotoxic and carcinogenic effects before being marketed to protect man and the environment from health risks. For agrochemicals, the long-term rodent carcinogenicity study is currently required from a regulatory perspective. Although it is the current mainstay for the detection of nongenotoxic carcinogens, carcinogenicity studies are shown to have prominent weaknesses and are subject to ethical and scientific debate. A transition toward a mechanism-based weight-of-evidence approach is considered a requirement to enhance the prediction of carcinogenic potential for environmental (agro)chemicals. The resulting approach should make optimal use of innovative (computational) tools and be less animal demanding. To identify the various mode of actions (MOAs) underlying the nongenotoxic carcinogenic potential of agrochemicals, we conducted an extensive analysis of 411 unique agrochemicals that have been evaluated for carcinogenicity by the United States Environmental Protection Agency (US EPA) and the European Chemicals Agency (ECHA). About one-third of these substances could be categorized as nongenotoxic carcinogens with an average of approximately two tumor types per substance, observed in a variety of organs. For two-third of the tumor cases, an underlying MOA (network) could be identified. This analysis demonstrates that a limited set of MOA (networks) is underlying nongenotoxic carcinogenicity of agrochemicals, illustrating that the transition toward a MOA-driven approach appears manageable. Ultimately the approach should cover relevant MOAs and its associated key events; this will also facilitate the evaluation of the human relevance. This manuscript describes the results of the analysis while identifying knowledge gaps and necessities to achieve a mechanism-based weight-of-evidence approach.

Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals.

Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.

Effects of agricultural pesticides on the reproductive system of aquatic wildlife species, with crocodilians as sentinel species.

Agricultural pesticides represent a significant class of endocrine-disrupting chemicals (EDCs) to which non-target organisms around the world are constantly exposed. Laboratory studies have found strong evidence showing the endocrine-disruptive potential of these pesticides at environmentally relevant exposure levels. Since the field of endocrine disruption continues to grow in richness and complexity, this review aims to provide an update on the effects of two agricultural pesticides that act as EDCs: atrazine and endosulfan. We will focus mainly on the effects on crocodilians due to their worldwide occurrence in tropical and sub-tropical wetland ecosystems and their ecological and physiological features, which render them vulnerable to exposure to pesticides with endocrine-disrupting action at all life stages. The results here reviewed provide important insights into the effects of hormonally active agricultural pesticides at cellular, tissue, and organ levels in the reproductive system of crocodiles. A better understanding of the effects of exposure to environmentally relevant doses of EDCs on the reproductive system of crocodilians will contribute to protect and improve the health of both wildlife species and humans.

Agrochemicals with estrogenic endocrine disrupting properties: Lessons Learned?

Many agrochemicals have endocrine disrupting properties. A subset of these chemicals is characterized as "estrogenic". In this review, we describe several distinct ways that chemicals used in crop production can affect estrogen signaling. Using three agrochemicals as examples (DDT, endosulfan, and atrazine), we illustrate how screening tests such as the US EPA's EDSP Tier 1 assays can be used as a first-pass approach to evaluate agrochemicals for endocrine activity. We then apply the "Key Characteristics" approach to illustrate how chemicals like DDT can be evaluated, together with the World Health Organization's definition of an endocrine disruptor, to identify data gaps. We conclude by describing important issues that must be addressed in the evaluation and regulation of hormonally active agrochemicals including mixture effects, efforts to reduce vertebrate animal use, chemical prioritization, and improvements in hazard, exposure, and risk assessments.

Relationship between agrochemical compounds and mammary gland development and breast cancer.

The exposure to agrochemical pesticides has been associated with several chronic diseases, including different types of cancer and reproductive disorders. In addition, because agrochemical pesticides may act as endocrine disrupting chemicals (EDCs) during different windows of susceptibility, they can increase the risk of impairing the normal development of the mammary gland and/or of developing mammary lesions. Therefore, the aim of this review is to summarize how exposure to different agrochemical pesticides suspected of being EDCs can interfere with the normal development of the mammary gland and the possible association with breast cancer. It has been shown that the mammary glands of male and female rats and mice are susceptible to exposure to non-organochlorine (vinclozolin, atrazine, glyphosate, chlorpyrifos) and organochlorine (endosulfan, methoxychlor, hexachlorobenzene) pesticides. Some of the effects of these compounds in experimental models include increased or decreased mammary development, impaired cell proliferation and steroid receptor expression and signaling, increased malignant cellular transformation and tumor development and angiogenesis. Contradictory findings have been found as to whether there is a causal link between the exposure or the pesticide body burden and breast cancer in humans. However, an association has been observed between pesticides (especially organochlorine compounds) and specific subtypes of breast cancer. Further studies are needed in both humans and experimental models to understand how agrochemical pesticides can induce or promote changes in the development, differentiation and/or malignant transformation of the mammary gland.

Organochlorine pesticides: Agrochemicals with potent endocrine-disrupting properties in fish.

Organochlorine pesticides (OCPs) are persistent environmental contaminants that act as endocrine disruptors and organ system toxicants. These pesticides (e.g. dichlorodiphenyltrichloroethane (DDT), dieldrin, toxaphene, among others) are ranked as some of the most concerning chemicals for human health. These pesticides (1) act as teratogens, (2) are neuroendocrine disruptors, (3) suppress the immune and reproductive systems, and (4) dysregulate lipids and metabolism. Using a computational approach, we revealed enriched endocrine-related pathways in the Comparative Toxicogenomics Database sensitive to this chemical class, and these included reproduction (gonadotropins, estradiol, androgen, steroid biosynthesis, oxytocin), thyroid hormone, and insulin. Insight from the Tox21 and ToxCast programs confirm that these agrochemicals activate estrogen receptors, androgen receptors, and retinoic acid receptors with relatively high affinity, although differences exist in their potency. We propose an adverse outcome pathway for OCPs toxicity in the fish testis as a novel contribution to further understanding of OCP-induced toxicity. Organochlorine pesticides, due to their persistence and high toxicity to aquatic and terrestrial wildlife as well as humans, remain significant agrochemicals of concern.