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Nutrigenomics of Food Pesticides
C H A P T E R
67 Nutrigenomics of Food Pesticides Laura Bordoni, Rosita Gabbianelli Unit of Molecular Biology, School of Pharmacy, University of Camerino, Camerino, Italy
Glossary 3-PBA 3-Phenoxybenzoic acid is the biomarkers of pyrethroid pesticide exposure discoverable in the people’’s urine. ADI Acceptable Daily Intake is “defined as the amount of specific substance that can be ingested on a daily basis without an appreciable health risk” (WHO, 1987). CYP Cytochrome P450 describes a group of enzymes whose main function is the oxidative catalysis of several exogenous and endogenous substances. GST Glutathione S-transferase catalyzes the reaction of glutathione with electrophiles of both endogenous and xenobiotic origins contributing to detoxification and protection against oxidative stress. HI The Hazard Index is used to assess the cumulative chronic health risk offor exposure to a group of pesticides; it defines the consumer risk in food. LEARn Latent Early life Associated Regulation. MRLs Maximum residue levels are the upper levels of pesticide residues that are legally permissible in food or animal feed, based on good agricultural practice and the lowest consumer exposure necessary to protect vulnerable consumers. NOAEL No Observed Adverse Effect Level is the dose that gives no toxic effect. It is extrapolated by chronic neurotoxicity studies done performed with animals. PON Paraoxonase is a group of proteins present in three forms (PON1, PON2, and PON3) encoded by genes PON1, PON2, and PON3. PUFAs Polyunsaturated fatty acids.
INTRODUCTION Pesticides are chemicals used to protect crops against noxious or unwanted living species such as insects, weeds, fungi, and other pests; they have a significant role in food production. Pesticides protect or increase yields and the number of times per year a crop can be grown on the same land, which is particularly important in countries facing food shortages. Nevertheless, pesticides have caused increasing concerns for human health, and their potentially harmful effects have been extensively studied over past decades. Both animal models of pesticide toxicity and observational studies on Principles of Nutrigenetics and Nutrigenomics https://doi.org/10.1016/B978-0-12-804572-5.00067-7
workers exposed to pesticides revealed that these compounds can be responsible for adverse effects. The acute toxicity of pesticides is well-known and their use is regulated on the basis of maximum residue levels (MRLs), which are established by considering, among others, the acceptable daily intake (ADI) for any particular pesticide in humans. Nevertheless, uncertainties remain concerning the chronic and long-term effects of exposure to pesticides. Delayed health effects attributed to pesticide exposure range from general malaise to chronic and longterm severe effects on the nervous system, including cognitive and psychomotor dysfunctions, mild cognitive dysfunctions (e.g., mood changes, neurobehavioral changes), depression, minor psychiatric morbidity, neurodegenerative (e.g., Parkinson and Alzheimer diseases), and neurodevelopmental effects. Reproductive functions can also be affected, with impaired fecundity, infertility, birth defects, and altered growth. Exposure to deceptively subtoxic doses of pesticides during a developmental period such as early life is a critical time window of particular concern. Numerous studies showed that developing fetuses, newborns, infants, and developed children are highly susceptible to late functional toxicity, which becomes manifest in adult life as the result of early-life exposure. In particular, developmental functional toxicity may affect the central nervous system, but effects have also been observed on the reproductive, endocrine, and immune systems. These observations suggest that professionals should know and consider these aspects to preserve health through good nutritional practices.
NONNUTRITIVE CHEMICALS IN FOODS Organizations in charge of food safety across the world constantly analyze levels of chemical residues (i.e., pesticides, insecticides, fungicides, herbicides) in
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Copyright © 2020 Elsevier Inc. All rights reserved.
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foods. The United States, Peoples’ Republic of China, and European Union have different laws and screening programs aimed at controlling and defining acceptable levels of chemicals that should have no toxic effects. A report on pesticide residues in 5989 samples of foods published by the US Food and Drug Administration stated that over 98% of domestic and 90% of imported foods were compliant with federal standards. No pesticide chemical residues were identified in 49.8% of domestic and 56.8% of imported human food samples analyzed (https://www.fda.gov/downloads/Food/ FoodborneIllnessContaminants/Pesticides/UCM582721. pdf). However, the ToxCast high-throughput screening program discovered that foods in the United States contained several chemical compounds that can be classified into four groups: chemicals added to foods for functional purposes (556 direct food additives); chemicals that may migrate to food from packaging, processing, or clinical chemicals (339 indirect additives); pesticide residues (406 compounds); and nonfood (319 compounds) (Karmaus et al., 2017). A list of these compounds is available online at http://ilsina.org/curationof-food-relevant-chemicals-in-toxcast. Use of these compounds is allowed by the Food and Drug Administration even though about 70% of these additives have no experimental data to clearly demonstrate their safety for human health. Chemicals included in the ToxCast classification were analyzed for their capacity to induce various effects, such as interactions with receptors, enzyme inhibition, induction of stress, and cytotoxicity. Within the pesticide group, all pesticide residues registered for food use in the United States, as well as pesticides not included in the accepted list in the United States but used elsewhere, were included. Of particular note is that among the nonfood group, 219 compounds were detected and recognized as pesticides not actually been demonstrated as being toxic, or that have nonfood crop uses. Among the nonfood compounds, sunscreen ingredients, insect repellents, anticoagulants, and antineoplastic and antipsychotic drugs have been identified. The European Food Safety Authority evaluated the presence of pesticides in food within MRLs allowed in European Union legislation. The European Food Safety Authority report revealed that more than 97% of foods analyzed in Europe contain the legal level of pesticide residues, 96.5% of foods for infants and young children were free of residues, and 99.3% of organic foods were residue-free or within the permitted limits (https:// www.efsa.europa.eu). Currently, 385 substances are authorized in the European Union, 26 of which are also accepted in organic agriculture. The risk assessment for food pesticides should consider not only the presence of chemicals above the authorized limit but also the presence of multiple kinds of pesticides, each within the MRLs permitted by the
legislation. In this case, the amplified effect of a mixture of pesticides should be considered, because the final biological effect is likely to reflect the sum of all chemical residues contained in food. Of particular relevance is the observation that many pesticide metabolites are as active as their parental compound and that the same class of pesticides results in the same kind of metabolites. Another key point for the controlled assessment of pesticide residues in food is related to the analytical method used in their extraction and detection, which can lead to significant differences in the evaluation of MRLs. The analytical recovery and final quantification can vary by 80% of the total, depending on the solvent used (i.e., ethyl acetate, dichloromethane, cyclohexane). In Asia, the wide presence of pesticides in food is of concern, as demonstrated by the significant increase in published articles on this topic in past couple of decades. The governing bodies are working to inform farmers about the need to regulate the quantity and quality of chemicals required to control crop production. As reported in samples collected in other geographic areas, chemicals can also be identified in organic products: 15 different pesticides were identified in samples of cabbage, celery, lettuce, broccoli, mustard, spinach, and cauliflower from organic farms in the Cameron Highlands, Malaysia (Farina et al., 2017). Among these vegetables, samples without detectable residues ranged from 6.6% to 30%, samples containing residues less than or at MRLs ranged between 60% and 90%, whereas 4% e13.3% contained resides at MRLs or higher. Only one sample among the 109 vegetables analyzed contained no residue over the MRLs. The Hazard Index (HI) has been used to assess the cumulative chronic health risk related to the exposure to a group of pesticides contained in foods for humans. This index is useful in screening the content of chemical residues in organic, conventional, and imported foods, and can provide a quantification of residues in different diets even when the pesticide level is below the no observed adverse effect level (NOAEL). The HI has been used to predict the cumulative effects of pesticides with different target organs. Although HI is useful for monitoring food quality, screening of biological markers of exposure is a major goal of health investigations in this area, because the metabolite residues of chemicals in urine, plasma, and blood adequately describe individual metabolic responses to pesticides according to one’s own genome.
BIOMARKERS OF PESTICIDE EXPOSURE IN HUMANS Biomarkers of pesticide exposure are mainly discoverable in the urine (McKelvey et al., 2013). For example,
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INDIVIDUALS’ RESPONSES TO PESTICIDES
the wide use of pyrethroids is demonstrated by the presence of 3-phenoxybenzoic acid (3-PBA) in people’s urine in China, the European Union, and the United States. The concern is linked to the observation that this metabolite is higher in children than in adults. Pyrethroids and their common metabolite, 3-PBA, can cross the bloode brain barrier and, owing to their lipophilicity, can be stored in the long term in the brain. Together with pyrethroids, organophosphates are the most common pesticides used in Europe. Their presence can be monitored in people’s urine owing to the presence of the dialkyl phosphate metabolites. Of particular concern are data obtained from the Center for the Health Assessment of Mothers and Children of Salinas cohort in the United States regarding the association between increased levels of organophosphate exposure biomarkers in the urine of farm worker mothers and abnormal mental development in their children. Dialkyl phosphates and 3-PBA were increased in children’s urine affected by attention-deficit hyperactive disorder; the risk for developing attention-deficit hyperactive disorder increased by 55% with a 10-fold increase in urine dialkyl phosphates, and the risk was twice as high in children with detectable concentrations of 3-PBA. A metaanalysis of articles published between 1979 and 2016 supported an association between residential exposure to pesticides and the development of childhood brain cancer. The Endocrine Disruptors : Longitudinal Study on Pregnancy Anomalies, Infertility, and Childhood cohort in France associated urine 3-PBA levels with defects in verbal and memory functions in 6-year-old children. The social cost of this decrease in intelligence quotient levels in children exposed to organophosphates has been estimated at about V125 billion. Constructive criticism underlines the relevance of statistical heterogeneity in these results, suggesting that further strategies are mandatory to improve knowledge regarding the link between markers of exposure and health effects. Furthermore, because of the various types and toxicity of pesticides, the development of assays aimed at identifying the cumulative risk for dietary chemical exposure in biological fluids (i.e., urine, plasma) should be a priority to establish the link between biomarkers of chemical exposure and diseases.
INDIVIDUALS’ RESPONSES TO PESTICIDES Exposure to the same environment does not always have the same effect on different subjects. On the contrary, important interindividual variability has emerged in responses to chemicals. Genetic differences in food tolerance, taste preferences, nutrient absorption, and metabolism all potentially affect the influence of diet
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on disease risk. Dietary constituents of foods, including both micronutrients and non-nutrients, act as inducing agents of metabolizing enzyme systems through several molecular mechanisms. In addition to direct dietary inducers, several nutritional factors (i.e., protein intake, obesity, fasting) have been shown to influence the activity of oxidation enzymes. Because diet is a combination of mutagens and protective agents that are all metabolized by detoxification enzymes, genetic variations in their gene structure, considered in the context of differential environmental exposures, may account for individual variation in disease risk. The identification of single nucleotide polymorphisms in human populations exposed to different environmental hazards has an essential role in detecting the genetic risks for inducing several important human diseases. For this purpose, numerous studies identified an association between single nucleotide polymorphisms in family of genes, such as those of paraoxonase (PON), cytochrome P450 (CYP), or glutathione S-transferases (GSTs), and pathologic conditions related to long-term exposure to pesticides (Lampe, 2007). For example, genotypes and consequent enzyme activities of human PON1 are associated with increased susceptibility to pesticide-related damage and oxidative stress-related health conditions (e.g., neurodegenerative disorders, cardiovascular diseases, diabetes, and obesity). In particular, several studies suggested the existence of a relation between PON1 polymorphisms and low PON1 protein levels as well as adverse developmental and cognitive behavior in children. Moreover, it has been shown that progeny of mothers with susceptible genotypes are more prone to toxicity related to prenatal exposure to organophosphates. CYP and N-acetyltransferase-2 are other xenobioticmetabolizing enzyme systems, the genetics of which has been studied for susceptibility to DNA damage resulting from pesticide exposure. DNA damage in workers exposed to organophosphate pesticides was higher in people with one particular allele compared with those with a different allele. Studies have shown associations between CYP1A1-PON1 polymorphisms and several diseases in individuals occupationally exposed to polychlorinated biphenyls. GSTs are other enzymes involved in detoxification processes. Prenatal exposure to pesticides in children of mothers with GSTM1 and GSTT1 alleles, which result in a lack of GSTM1 and GSTT1 proteins, respectively, exposes them to a greater risk for fetal growth restriction. Furthermore, polymorphisms in the GSTM1 and GSTT1 genes were associated with a higher risk for cancer, which, by contrast, is strongly reduced with the intake of cruciferous vegetables. Thus, it is clear that an interaction exists between susceptibility to xenobiotics and diet, and that further knowledge of
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susceptibility based on genetic factors could target dietary interventions for individuals most likely to benefit.
MODULATION BY PESTICIDES OF GENE EXPRESSION AND EPIGENETICS Food pesticides contribute to the human exposome, which is one of the most promising research areas for the therapy and prevention of disease. The exposome is defined as “the cumulative measure of environmental influences and associated biological responses throughout the life span, including exposures to the environment, diet, behavior, and endogenous processes” (Dennis and Jones, 2016). Thus, exposure to contaminants is emerging as related to the contaminant content in foods and as having a strong impact on health: the presence of certain chemicals per se has a critical role in health, but also how those molecules alter our biology and how resilient the organism is in maintaining dynamic homeostasis. Environmental exposure can be responsible for several potential effects, such as DNA mutation, adducts, epigenetic changes or alterations in enzyme functions, or damage induced through reactive oxygen or nitrogen species. Furthermore, environmental exposure can cause structural changes as well as changes in gene expression profile in exposed tissues. Numerous studies showed that lipophilic pesticides can accumulate in adipose tissue, exerting an obesogenic effect through subtle changes in gene expression and tissue organization. Transcriptomic analysis also revealed changes in gene expression in animal brains exposed to pesticides, and linked changes in the expression patterns of messenger RNAs with neurodegeneration induced by the exposure. Moreover, besides adductomic and metabolomic approaches, increasing importance is being attributed to evidence in the literature that environmental factors may cause diseases through changes in gene expression, in turn mediated by epigenetic mechanisms such as DNA methylation, histone modifications, and chromatin remodeling, all of which are potentially implicated in disease triggering induced by numerous environmental factors (Collotta et al., 2013). A few examples are exposure in early life to subtle concentration of permethrin, which can lead to global DNA methylation changes; levels of persistent organic pollutants in the blood, which were inversely associated with global DNA methylation; exposure to dichlorodiphenyltrichloroethane, which was linked to altered DNA methylation patterns in the rat hypothalamus; paraquat, which can induce histone H3 acetylation and decrease histone deacetylase activity; and dieldrin-induced proteasomal dysfunction, which can result in the accumulation of
histone acetyltransferases. Whenever exposure to dieldrin is prolonged, it induces histone hyperacetylation in the corpus striatum and substantia nigra in mice. Furthermore, among many other factors, both genetic and epigenetic determinants contribute to the final biological response. PON1 expression is an example of how both of these aspects regulate responses to xenobiotics. Even when PON1 expression is strongly regulated by genetics (see the previous discussion), other factors such as epigenetic changes are involved in controlling PON1 enzyme variability. This occurs because the PON1108 promoter polymorphism is associated with methylation of particular CpG sites, which suggests that DNA methylation can be mediate PON1 genetics and its expression. These findings are an example of how the integration of genetic, epigenetic, and expression data could clarify functional mechanisms involved in susceptibility to xenobiotics, and that the totality of genetic background and environmental exposures has to be considered in evaluating disease prevention strategies at the individual and population levels (Declerck et al., 2017). Advances in transcriptomics, genomics, and epigenomics thus provide insights into susceptibility to diseases, increasing our understanding of the molecular mechanisms behind their onset. Furthermore, these advances help to identify new kinds of biomarkers, particularly referring to epigenetic changes, and provide information about exposures, but also biological effects, thus offering an important set of tools in the area of personalized medicine and prevention.
PESTICIDES AS EARLY DETERMINANTS OF LATE-ONSET DISEASES Because of all of the previously listed complex molecular mechanisms through which pesticides can interact (including those contained in foods), numerous diseases have been related to exposure to environmental toxicants. Particularly when taking place in the specific window of plasticity, they may contribute to the occurrence of adverse birth outcomes, neurodevelopmental deficits, increased risk for cancer, and other multifactorial diseases such as asthma and diabetes. Furthermore, not only the generation directly exposed but also its offspring can be affected through mechanisms of epigenetic memory. These revolutionary discoveries that have emerged from both epidemiological and in vivo studies emphasize the importance of considering the subtle effect of pesticides on health. For example, the epigenetic effect of bisphenol A was originally shown in viable yellow mice by decreasing CpG methylation upstream of the Agouti gene, and maternal diet supplementation with methyl donors such as folic acid or the phytoestrogen genistein, was shown to prevent the
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NUTRIGENOMICS AS A STRATEGY FOR BIOREMEDIATION
hypomethylating effect of the toxicant. In addition, epigenetic effects of bisphenol A and phthalates were also demonstrated and linked to complex diseases such as cancer and diabetes in humans. Moreover, pesticides such as dieldrin, paraquat, maneb, and permethrin were shown to lead to Parkinson-like disease through dysfunction of the nigrostriatal dopaminergic system and/or abnormalities in motor response in animal studies, which proves that early-life exposure to these chemicals can exert adverse effects later in life. Furthermore, Skinner and colleagues demonstrated that exposure of gestating rats to methoxychlor increases the incidence of kidney disease, ovary disease, and obesity in offspring, spanning three generations. Thus, nutrition can influence epigenetic homeostasis by providing substrates, cofactors for epigenetic reactions, and epigenetically active molecules, but also by being a source of exposure to exogenous molecules able to interfere with this equilibrium (Skinner, 2016). Several different models have been hypothesized to explain the role of epigenetics in disease onset. Barker hypothesized that adult diseases are consequences of fetal adverse conditions, because the fetus adapts to a certain environment determined by environmental stimuli in early life. Adaptive responses may be in the form of metabolic changes, hormonal release, or sensitivity of the target organs to hormones, all of which then affect the development of target organs with no immediate consequences to the newborn, but with later disturbances in physiologic and metabolic functions. Gluckman and Hanson suggested that when the fetus is exposed to stress or adverse conditions, immediate reversible changes occur. However, when stress conditions are prolonged, changes become irreversible and able to persist throughout life, influencing the individual persistently in adulthood. They defined this phenomenon using the term “predictive adaptive response.” Effects of these irreversible changes on the fetus in the long run may be favorable or unfavorable. The Developmental Origin of Health and Disease model postulated that not only embryonic stages but also the period of development during infancy is responsible for the late-life risk for disease. A further evolution of these models aiming to explain the role of environmental factors in disease etiology, with a particular focus on neurodegeneration is represented by the Latent Early life Associated Regulation (LEARn) model. This theory states that environmental agents, including nutrition, metal exposure, head traumas, and lifestyle, are hits related to the cause and progression of sporadic neurodegenerative disease onset. LEARn differs from other models in that it is neither an acute nor a chronic model, but is based on the idea that latent epigenetic changes induced in early life, which result in no disease symptoms, create a perturbation in the epigenome, causing
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manifest consequences later in life after the occurrence of a second triggering agent. The period between the epigenetic perturbation and the second triggering event is called the latency period, and genes that respond late in relation to early-life responses are called LEARned genes. The process of responding to the early-life environmental triggers after the long latency period is termed LEARning.
NUTRIGENOMICS AS A STRATEGY FOR BIOREMEDIATION The sensitivity of populations to the consequences of food pesticides is increasing, as demonstrated by the growth of the organic retail market by 107% between 2006 and 2015 in the European Union; it reached a total of V27 billion. Generally, people consuming organic food have a healthy lifestyle, and their food is rich in fresh fruits and vegetables useful for the presence of bioactive compounds able to modulate gene expression beneficially. A metaanalysis of 343 peer-reviewed publications underlined the presence of higher levels of antioxidants and lower concentrations of cadmium in organically grown crops compared with conventionally grown ones (Bara nski et al., 2014). A metaanalysis of 170 articles related to organic milk composition revealed that polyunsaturated fatty acids (PUFAs), specifically n-3 PUFAs, were significantly higher, by 7% and 56%, respectively, compared with conventional milk, whereas no change in saturated fatty acids and monounsaturated fatty acids was observed. Furthermore, the concentrations of conjugated linoleic acid, a-tocopherol, and iron increased. Another metaanalysis of 67 articles highlighted an increase in PUFA and n-3 PUFA, by 23% and 47%, respectively, in organic meat compared with conventional meat, which underlines that these parameters strongly depend on differences among animal species providing the source of meat. The consumption of organic food is associated with a lower occurrence of allergy and atopic diseases in children. In particular, a reduction by 36% in the risk for eczema in 2-year-old infants was demonstrated when their mothers (n ¼ 2700) consumed organic food during pregnancy and breastfeeding. A study of 62,000 participants consuming organic food demonstrated a 31% decrease in body mass index compared with those who consumed conventional foods. Another study among 630,080 middle-aged United Kingdom women reported a reduction in the risk for non-Hodgkin lymphoma in subjects consuming more organic foods compared with the control group. The use of HI in Sweden was proposed to quantify the exposure to toxicants owing to the consumption of 500 g fruits and vegetables divided into three groups: imported conventional,
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domestic conventional, or organically produced foods. The HI indicated that exposure to toxicants was about 70 times lower when organic food was consumed. According to this evidence, organic food may be a strategy for increasing the level of bioactive compounds able to modulate gene expression favorably and also reduce the exposure to pesticides. An alternative strategy might be to consider using selected bioactive food or supplements; attention should be given to the molecular mechanisms of pesticide damage. To partially counterbalance adverse effects, dietary agents with antioxidant and antiinflammatory properties, as well as bioactive components able to mediate epigenetic modifications, might be useful. As reported in experimental animal models, coadministration of low-dose pesticides and antioxidants significantly decreased parameters associated with oxidative stress caused by pesticide exposure. In particular, the moderate use of vitamin C, glutathione, and vitamin E can favorably modulate the expression or activity of transcription factors such as nuclear factor-kB, Nrf2, and cytokines. Green tea polyphenols, black raspberries, soy isoflavones, curcumin, apple/coffee polyphenols, broccoli isothiocyanates, vitamin A, selenium, lycopene, folate, and vitamins B12 and B6 work as preventative agents modulating DNA methylation and preventing aberrant promoter hypermethylation or genome-wide hypomethylation for cancer and chronic diseases. Sulforaphane from broccoli, diallyl disulfide from garlic, and butyrate from fiber by gut microbiota are histone deacetylase inhibitors useful for controlling inflammation. Studies show that consumption of 105 mg sulforaphane contained in 68 g broccoli sprouts or 570 g mature broccoli can significantly inhibit histone deacetylase activity after 3 h. Sulforaphane has also been demonstrated to be useful in promoting detoxifying enzymes required to metabolize pesticides.
Despite these outcomes, additional epidemiological studies are required on the efficacy of organic food or supplements to contrast pesticide-induced damage, together with an evaluation of pesticide residue metabolites in biological fluids that provide data on human exposure and the individual genetic capacity to metabolize these chemicals.
References Bara nski, M., Srednicka-Tober, D., Volakakis, N., Seal, C., Sanderson, R., Stewart, G.B., Benbrook, C., Biavati, B., Markellou, E., Giotis, C., Gromadzka-Ostrowska, J., Rembiałkowska, E., Skwarło-So nta, K., Tahvonen, R., Janovska´, D., Niggli, U., Nicot, P., Leifert, C., 2014. Higher antioxidant and lower cadmium concentrations and lower incidence of pesticide residues in organically grown crops: a systematic literature review and meta-analyses. Br J Nutr 112 (5), 794e811. Collotta, M., Bertazzi, P.A., Bollati, V., 2013. Epigenetics and pesticides. Toxicology 307, 35e41. Declerck, K., Remy, S., Wohlfahrt-Veje, C., Main, K.M., Van Camp, G., Schoeters, G., Vanden Berghe, W., Andersen, H.R., 2017. Interaction between prenatal pesticide exposure and a common polymorphism in the PON1 gene on DNA methylation in genes associated with cardio-metabolic disease risk-an exploratory study. Clin Epigenetics 9, 35. Dennis, K.K., Jones, D.P., 2016. The exposome: a new frontier for education. Am Biol Teach 78, 542e548. Farina, Y., Abdullah, M.P., Bibi, N., Khalik, W.M., 2017. Determination of pesticide residues in leafy vegetables at parts per billion levels by a chemometric study using GC-ECD in Cameron Highlands, Malaysia. Food Chem 224, 55e61. Karmaus, A.L., Trautman, T.D., Krishan, M., Filer, D.L., Fix, L.A., 2017. Curation of food-relevant chemicals in ToxCast. Food Chem Toxicol 103, 174e182. Lampe, J.W., 2007. Diet, genetic polymorphisms, detoxification, and health risks. Altern Ther Health Med 13 (2), S108eS111. McKelvey, W., Jacobson, J.B., Kass, D., Barr, D.B., Davis, M., Calafat, A.M., Aldous, K.M., 2013. Population-based biomonitoring of exposure to organophosphate and pyrethroid pesticides in New York city. Environ Health Perspect 121 (11e12), 1349e1356. Skinner, M.K., 2016. Endocrine disruptors in 2015: epigenetic transgenerational inheritance. Nat Rev Endocrinol 12 (2), 68e70.
IV. TRANSLATIONAL NUTRIGENETICS AND NUTRIGENOMICS
67 Nutrigenomics of Food Pesticides Laura Bordoni, Rosita Gabbianelli Unit of Molecular Biology, School of Pharmacy, University of Camerino, Camerino, Italy
Glossary 3-PBA 3-Phenoxybenzoic acid is the biomarkers of pyrethroid pesticide exposure discoverable in the people’’s urine. ADI Acceptable Daily Intake is “defined as the amount of specific substance that can be ingested on a daily basis without an appreciable health risk” (WHO, 1987). CYP Cytochrome P450 describes a group of enzymes whose main function is the oxidative catalysis of several exogenous and endogenous substances. GST Glutathione S-transferase catalyzes the reaction of glutathione with electrophiles of both endogenous and xenobiotic origins contributing to detoxification and protection against oxidative stress. HI The Hazard Index is used to assess the cumulative chronic health risk offor exposure to a group of pesticides; it defines the consumer risk in food. LEARn Latent Early life Associated Regulation. MRLs Maximum residue levels are the upper levels of pesticide residues that are legally permissible in food or animal feed, based on good agricultural practice and the lowest consumer exposure necessary to protect vulnerable consumers. NOAEL No Observed Adverse Effect Level is the dose that gives no toxic effect. It is extrapolated by chronic neurotoxicity studies done performed with animals. PON Paraoxonase is a group of proteins present in three forms (PON1, PON2, and PON3) encoded by genes PON1, PON2, and PON3. PUFAs Polyunsaturated fatty acids.
INTRODUCTION Pesticides are chemicals used to protect crops against noxious or unwanted living species such as insects, weeds, fungi, and other pests; they have a significant role in food production. Pesticides protect or increase yields and the number of times per year a crop can be grown on the same land, which is particularly important in countries facing food shortages. Nevertheless, pesticides have caused increasing concerns for human health, and their potentially harmful effects have been extensively studied over past decades. Both animal models of pesticide toxicity and observational studies on Principles of Nutrigenetics and Nutrigenomics https://doi.org/10.1016/B978-0-12-804572-5.00067-7
workers exposed to pesticides revealed that these compounds can be responsible for adverse effects. The acute toxicity of pesticides is well-known and their use is regulated on the basis of maximum residue levels (MRLs), which are established by considering, among others, the acceptable daily intake (ADI) for any particular pesticide in humans. Nevertheless, uncertainties remain concerning the chronic and long-term effects of exposure to pesticides. Delayed health effects attributed to pesticide exposure range from general malaise to chronic and longterm severe effects on the nervous system, including cognitive and psychomotor dysfunctions, mild cognitive dysfunctions (e.g., mood changes, neurobehavioral changes), depression, minor psychiatric morbidity, neurodegenerative (e.g., Parkinson and Alzheimer diseases), and neurodevelopmental effects. Reproductive functions can also be affected, with impaired fecundity, infertility, birth defects, and altered growth. Exposure to deceptively subtoxic doses of pesticides during a developmental period such as early life is a critical time window of particular concern. Numerous studies showed that developing fetuses, newborns, infants, and developed children are highly susceptible to late functional toxicity, which becomes manifest in adult life as the result of early-life exposure. In particular, developmental functional toxicity may affect the central nervous system, but effects have also been observed on the reproductive, endocrine, and immune systems. These observations suggest that professionals should know and consider these aspects to preserve health through good nutritional practices.
NONNUTRITIVE CHEMICALS IN FOODS Organizations in charge of food safety across the world constantly analyze levels of chemical residues (i.e., pesticides, insecticides, fungicides, herbicides) in
513
Copyright © 2020 Elsevier Inc. All rights reserved.
514
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foods. The United States, Peoples’ Republic of China, and European Union have different laws and screening programs aimed at controlling and defining acceptable levels of chemicals that should have no toxic effects. A report on pesticide residues in 5989 samples of foods published by the US Food and Drug Administration stated that over 98% of domestic and 90% of imported foods were compliant with federal standards. No pesticide chemical residues were identified in 49.8% of domestic and 56.8% of imported human food samples analyzed (https://www.fda.gov/downloads/Food/ FoodborneIllnessContaminants/Pesticides/UCM582721. pdf). However, the ToxCast high-throughput screening program discovered that foods in the United States contained several chemical compounds that can be classified into four groups: chemicals added to foods for functional purposes (556 direct food additives); chemicals that may migrate to food from packaging, processing, or clinical chemicals (339 indirect additives); pesticide residues (406 compounds); and nonfood (319 compounds) (Karmaus et al., 2017). A list of these compounds is available online at http://ilsina.org/curationof-food-relevant-chemicals-in-toxcast. Use of these compounds is allowed by the Food and Drug Administration even though about 70% of these additives have no experimental data to clearly demonstrate their safety for human health. Chemicals included in the ToxCast classification were analyzed for their capacity to induce various effects, such as interactions with receptors, enzyme inhibition, induction of stress, and cytotoxicity. Within the pesticide group, all pesticide residues registered for food use in the United States, as well as pesticides not included in the accepted list in the United States but used elsewhere, were included. Of particular note is that among the nonfood group, 219 compounds were detected and recognized as pesticides not actually been demonstrated as being toxic, or that have nonfood crop uses. Among the nonfood compounds, sunscreen ingredients, insect repellents, anticoagulants, and antineoplastic and antipsychotic drugs have been identified. The European Food Safety Authority evaluated the presence of pesticides in food within MRLs allowed in European Union legislation. The European Food Safety Authority report revealed that more than 97% of foods analyzed in Europe contain the legal level of pesticide residues, 96.5% of foods for infants and young children were free of residues, and 99.3% of organic foods were residue-free or within the permitted limits (https:// www.efsa.europa.eu). Currently, 385 substances are authorized in the European Union, 26 of which are also accepted in organic agriculture. The risk assessment for food pesticides should consider not only the presence of chemicals above the authorized limit but also the presence of multiple kinds of pesticides, each within the MRLs permitted by the
legislation. In this case, the amplified effect of a mixture of pesticides should be considered, because the final biological effect is likely to reflect the sum of all chemical residues contained in food. Of particular relevance is the observation that many pesticide metabolites are as active as their parental compound and that the same class of pesticides results in the same kind of metabolites. Another key point for the controlled assessment of pesticide residues in food is related to the analytical method used in their extraction and detection, which can lead to significant differences in the evaluation of MRLs. The analytical recovery and final quantification can vary by 80% of the total, depending on the solvent used (i.e., ethyl acetate, dichloromethane, cyclohexane). In Asia, the wide presence of pesticides in food is of concern, as demonstrated by the significant increase in published articles on this topic in past couple of decades. The governing bodies are working to inform farmers about the need to regulate the quantity and quality of chemicals required to control crop production. As reported in samples collected in other geographic areas, chemicals can also be identified in organic products: 15 different pesticides were identified in samples of cabbage, celery, lettuce, broccoli, mustard, spinach, and cauliflower from organic farms in the Cameron Highlands, Malaysia (Farina et al., 2017). Among these vegetables, samples without detectable residues ranged from 6.6% to 30%, samples containing residues less than or at MRLs ranged between 60% and 90%, whereas 4% e13.3% contained resides at MRLs or higher. Only one sample among the 109 vegetables analyzed contained no residue over the MRLs. The Hazard Index (HI) has been used to assess the cumulative chronic health risk related to the exposure to a group of pesticides contained in foods for humans. This index is useful in screening the content of chemical residues in organic, conventional, and imported foods, and can provide a quantification of residues in different diets even when the pesticide level is below the no observed adverse effect level (NOAEL). The HI has been used to predict the cumulative effects of pesticides with different target organs. Although HI is useful for monitoring food quality, screening of biological markers of exposure is a major goal of health investigations in this area, because the metabolite residues of chemicals in urine, plasma, and blood adequately describe individual metabolic responses to pesticides according to one’s own genome.
BIOMARKERS OF PESTICIDE EXPOSURE IN HUMANS Biomarkers of pesticide exposure are mainly discoverable in the urine (McKelvey et al., 2013). For example,
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the wide use of pyrethroids is demonstrated by the presence of 3-phenoxybenzoic acid (3-PBA) in people’s urine in China, the European Union, and the United States. The concern is linked to the observation that this metabolite is higher in children than in adults. Pyrethroids and their common metabolite, 3-PBA, can cross the bloode brain barrier and, owing to their lipophilicity, can be stored in the long term in the brain. Together with pyrethroids, organophosphates are the most common pesticides used in Europe. Their presence can be monitored in people’s urine owing to the presence of the dialkyl phosphate metabolites. Of particular concern are data obtained from the Center for the Health Assessment of Mothers and Children of Salinas cohort in the United States regarding the association between increased levels of organophosphate exposure biomarkers in the urine of farm worker mothers and abnormal mental development in their children. Dialkyl phosphates and 3-PBA were increased in children’s urine affected by attention-deficit hyperactive disorder; the risk for developing attention-deficit hyperactive disorder increased by 55% with a 10-fold increase in urine dialkyl phosphates, and the risk was twice as high in children with detectable concentrations of 3-PBA. A metaanalysis of articles published between 1979 and 2016 supported an association between residential exposure to pesticides and the development of childhood brain cancer. The Endocrine Disruptors : Longitudinal Study on Pregnancy Anomalies, Infertility, and Childhood cohort in France associated urine 3-PBA levels with defects in verbal and memory functions in 6-year-old children. The social cost of this decrease in intelligence quotient levels in children exposed to organophosphates has been estimated at about V125 billion. Constructive criticism underlines the relevance of statistical heterogeneity in these results, suggesting that further strategies are mandatory to improve knowledge regarding the link between markers of exposure and health effects. Furthermore, because of the various types and toxicity of pesticides, the development of assays aimed at identifying the cumulative risk for dietary chemical exposure in biological fluids (i.e., urine, plasma) should be a priority to establish the link between biomarkers of chemical exposure and diseases.
INDIVIDUALS’ RESPONSES TO PESTICIDES Exposure to the same environment does not always have the same effect on different subjects. On the contrary, important interindividual variability has emerged in responses to chemicals. Genetic differences in food tolerance, taste preferences, nutrient absorption, and metabolism all potentially affect the influence of diet
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on disease risk. Dietary constituents of foods, including both micronutrients and non-nutrients, act as inducing agents of metabolizing enzyme systems through several molecular mechanisms. In addition to direct dietary inducers, several nutritional factors (i.e., protein intake, obesity, fasting) have been shown to influence the activity of oxidation enzymes. Because diet is a combination of mutagens and protective agents that are all metabolized by detoxification enzymes, genetic variations in their gene structure, considered in the context of differential environmental exposures, may account for individual variation in disease risk. The identification of single nucleotide polymorphisms in human populations exposed to different environmental hazards has an essential role in detecting the genetic risks for inducing several important human diseases. For this purpose, numerous studies identified an association between single nucleotide polymorphisms in family of genes, such as those of paraoxonase (PON), cytochrome P450 (CYP), or glutathione S-transferases (GSTs), and pathologic conditions related to long-term exposure to pesticides (Lampe, 2007). For example, genotypes and consequent enzyme activities of human PON1 are associated with increased susceptibility to pesticide-related damage and oxidative stress-related health conditions (e.g., neurodegenerative disorders, cardiovascular diseases, diabetes, and obesity). In particular, several studies suggested the existence of a relation between PON1 polymorphisms and low PON1 protein levels as well as adverse developmental and cognitive behavior in children. Moreover, it has been shown that progeny of mothers with susceptible genotypes are more prone to toxicity related to prenatal exposure to organophosphates. CYP and N-acetyltransferase-2 are other xenobioticmetabolizing enzyme systems, the genetics of which has been studied for susceptibility to DNA damage resulting from pesticide exposure. DNA damage in workers exposed to organophosphate pesticides was higher in people with one particular allele compared with those with a different allele. Studies have shown associations between CYP1A1-PON1 polymorphisms and several diseases in individuals occupationally exposed to polychlorinated biphenyls. GSTs are other enzymes involved in detoxification processes. Prenatal exposure to pesticides in children of mothers with GSTM1 and GSTT1 alleles, which result in a lack of GSTM1 and GSTT1 proteins, respectively, exposes them to a greater risk for fetal growth restriction. Furthermore, polymorphisms in the GSTM1 and GSTT1 genes were associated with a higher risk for cancer, which, by contrast, is strongly reduced with the intake of cruciferous vegetables. Thus, it is clear that an interaction exists between susceptibility to xenobiotics and diet, and that further knowledge of
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susceptibility based on genetic factors could target dietary interventions for individuals most likely to benefit.
MODULATION BY PESTICIDES OF GENE EXPRESSION AND EPIGENETICS Food pesticides contribute to the human exposome, which is one of the most promising research areas for the therapy and prevention of disease. The exposome is defined as “the cumulative measure of environmental influences and associated biological responses throughout the life span, including exposures to the environment, diet, behavior, and endogenous processes” (Dennis and Jones, 2016). Thus, exposure to contaminants is emerging as related to the contaminant content in foods and as having a strong impact on health: the presence of certain chemicals per se has a critical role in health, but also how those molecules alter our biology and how resilient the organism is in maintaining dynamic homeostasis. Environmental exposure can be responsible for several potential effects, such as DNA mutation, adducts, epigenetic changes or alterations in enzyme functions, or damage induced through reactive oxygen or nitrogen species. Furthermore, environmental exposure can cause structural changes as well as changes in gene expression profile in exposed tissues. Numerous studies showed that lipophilic pesticides can accumulate in adipose tissue, exerting an obesogenic effect through subtle changes in gene expression and tissue organization. Transcriptomic analysis also revealed changes in gene expression in animal brains exposed to pesticides, and linked changes in the expression patterns of messenger RNAs with neurodegeneration induced by the exposure. Moreover, besides adductomic and metabolomic approaches, increasing importance is being attributed to evidence in the literature that environmental factors may cause diseases through changes in gene expression, in turn mediated by epigenetic mechanisms such as DNA methylation, histone modifications, and chromatin remodeling, all of which are potentially implicated in disease triggering induced by numerous environmental factors (Collotta et al., 2013). A few examples are exposure in early life to subtle concentration of permethrin, which can lead to global DNA methylation changes; levels of persistent organic pollutants in the blood, which were inversely associated with global DNA methylation; exposure to dichlorodiphenyltrichloroethane, which was linked to altered DNA methylation patterns in the rat hypothalamus; paraquat, which can induce histone H3 acetylation and decrease histone deacetylase activity; and dieldrin-induced proteasomal dysfunction, which can result in the accumulation of
histone acetyltransferases. Whenever exposure to dieldrin is prolonged, it induces histone hyperacetylation in the corpus striatum and substantia nigra in mice. Furthermore, among many other factors, both genetic and epigenetic determinants contribute to the final biological response. PON1 expression is an example of how both of these aspects regulate responses to xenobiotics. Even when PON1 expression is strongly regulated by genetics (see the previous discussion), other factors such as epigenetic changes are involved in controlling PON1 enzyme variability. This occurs because the PON1108 promoter polymorphism is associated with methylation of particular CpG sites, which suggests that DNA methylation can be mediate PON1 genetics and its expression. These findings are an example of how the integration of genetic, epigenetic, and expression data could clarify functional mechanisms involved in susceptibility to xenobiotics, and that the totality of genetic background and environmental exposures has to be considered in evaluating disease prevention strategies at the individual and population levels (Declerck et al., 2017). Advances in transcriptomics, genomics, and epigenomics thus provide insights into susceptibility to diseases, increasing our understanding of the molecular mechanisms behind their onset. Furthermore, these advances help to identify new kinds of biomarkers, particularly referring to epigenetic changes, and provide information about exposures, but also biological effects, thus offering an important set of tools in the area of personalized medicine and prevention.
PESTICIDES AS EARLY DETERMINANTS OF LATE-ONSET DISEASES Because of all of the previously listed complex molecular mechanisms through which pesticides can interact (including those contained in foods), numerous diseases have been related to exposure to environmental toxicants. Particularly when taking place in the specific window of plasticity, they may contribute to the occurrence of adverse birth outcomes, neurodevelopmental deficits, increased risk for cancer, and other multifactorial diseases such as asthma and diabetes. Furthermore, not only the generation directly exposed but also its offspring can be affected through mechanisms of epigenetic memory. These revolutionary discoveries that have emerged from both epidemiological and in vivo studies emphasize the importance of considering the subtle effect of pesticides on health. For example, the epigenetic effect of bisphenol A was originally shown in viable yellow mice by decreasing CpG methylation upstream of the Agouti gene, and maternal diet supplementation with methyl donors such as folic acid or the phytoestrogen genistein, was shown to prevent the
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hypomethylating effect of the toxicant. In addition, epigenetic effects of bisphenol A and phthalates were also demonstrated and linked to complex diseases such as cancer and diabetes in humans. Moreover, pesticides such as dieldrin, paraquat, maneb, and permethrin were shown to lead to Parkinson-like disease through dysfunction of the nigrostriatal dopaminergic system and/or abnormalities in motor response in animal studies, which proves that early-life exposure to these chemicals can exert adverse effects later in life. Furthermore, Skinner and colleagues demonstrated that exposure of gestating rats to methoxychlor increases the incidence of kidney disease, ovary disease, and obesity in offspring, spanning three generations. Thus, nutrition can influence epigenetic homeostasis by providing substrates, cofactors for epigenetic reactions, and epigenetically active molecules, but also by being a source of exposure to exogenous molecules able to interfere with this equilibrium (Skinner, 2016). Several different models have been hypothesized to explain the role of epigenetics in disease onset. Barker hypothesized that adult diseases are consequences of fetal adverse conditions, because the fetus adapts to a certain environment determined by environmental stimuli in early life. Adaptive responses may be in the form of metabolic changes, hormonal release, or sensitivity of the target organs to hormones, all of which then affect the development of target organs with no immediate consequences to the newborn, but with later disturbances in physiologic and metabolic functions. Gluckman and Hanson suggested that when the fetus is exposed to stress or adverse conditions, immediate reversible changes occur. However, when stress conditions are prolonged, changes become irreversible and able to persist throughout life, influencing the individual persistently in adulthood. They defined this phenomenon using the term “predictive adaptive response.” Effects of these irreversible changes on the fetus in the long run may be favorable or unfavorable. The Developmental Origin of Health and Disease model postulated that not only embryonic stages but also the period of development during infancy is responsible for the late-life risk for disease. A further evolution of these models aiming to explain the role of environmental factors in disease etiology, with a particular focus on neurodegeneration is represented by the Latent Early life Associated Regulation (LEARn) model. This theory states that environmental agents, including nutrition, metal exposure, head traumas, and lifestyle, are hits related to the cause and progression of sporadic neurodegenerative disease onset. LEARn differs from other models in that it is neither an acute nor a chronic model, but is based on the idea that latent epigenetic changes induced in early life, which result in no disease symptoms, create a perturbation in the epigenome, causing
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manifest consequences later in life after the occurrence of a second triggering agent. The period between the epigenetic perturbation and the second triggering event is called the latency period, and genes that respond late in relation to early-life responses are called LEARned genes. The process of responding to the early-life environmental triggers after the long latency period is termed LEARning.
NUTRIGENOMICS AS A STRATEGY FOR BIOREMEDIATION The sensitivity of populations to the consequences of food pesticides is increasing, as demonstrated by the growth of the organic retail market by 107% between 2006 and 2015 in the European Union; it reached a total of V27 billion. Generally, people consuming organic food have a healthy lifestyle, and their food is rich in fresh fruits and vegetables useful for the presence of bioactive compounds able to modulate gene expression beneficially. A metaanalysis of 343 peer-reviewed publications underlined the presence of higher levels of antioxidants and lower concentrations of cadmium in organically grown crops compared with conventionally grown ones (Bara nski et al., 2014). A metaanalysis of 170 articles related to organic milk composition revealed that polyunsaturated fatty acids (PUFAs), specifically n-3 PUFAs, were significantly higher, by 7% and 56%, respectively, compared with conventional milk, whereas no change in saturated fatty acids and monounsaturated fatty acids was observed. Furthermore, the concentrations of conjugated linoleic acid, a-tocopherol, and iron increased. Another metaanalysis of 67 articles highlighted an increase in PUFA and n-3 PUFA, by 23% and 47%, respectively, in organic meat compared with conventional meat, which underlines that these parameters strongly depend on differences among animal species providing the source of meat. The consumption of organic food is associated with a lower occurrence of allergy and atopic diseases in children. In particular, a reduction by 36% in the risk for eczema in 2-year-old infants was demonstrated when their mothers (n ¼ 2700) consumed organic food during pregnancy and breastfeeding. A study of 62,000 participants consuming organic food demonstrated a 31% decrease in body mass index compared with those who consumed conventional foods. Another study among 630,080 middle-aged United Kingdom women reported a reduction in the risk for non-Hodgkin lymphoma in subjects consuming more organic foods compared with the control group. The use of HI in Sweden was proposed to quantify the exposure to toxicants owing to the consumption of 500 g fruits and vegetables divided into three groups: imported conventional,
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domestic conventional, or organically produced foods. The HI indicated that exposure to toxicants was about 70 times lower when organic food was consumed. According to this evidence, organic food may be a strategy for increasing the level of bioactive compounds able to modulate gene expression favorably and also reduce the exposure to pesticides. An alternative strategy might be to consider using selected bioactive food or supplements; attention should be given to the molecular mechanisms of pesticide damage. To partially counterbalance adverse effects, dietary agents with antioxidant and antiinflammatory properties, as well as bioactive components able to mediate epigenetic modifications, might be useful. As reported in experimental animal models, coadministration of low-dose pesticides and antioxidants significantly decreased parameters associated with oxidative stress caused by pesticide exposure. In particular, the moderate use of vitamin C, glutathione, and vitamin E can favorably modulate the expression or activity of transcription factors such as nuclear factor-kB, Nrf2, and cytokines. Green tea polyphenols, black raspberries, soy isoflavones, curcumin, apple/coffee polyphenols, broccoli isothiocyanates, vitamin A, selenium, lycopene, folate, and vitamins B12 and B6 work as preventative agents modulating DNA methylation and preventing aberrant promoter hypermethylation or genome-wide hypomethylation for cancer and chronic diseases. Sulforaphane from broccoli, diallyl disulfide from garlic, and butyrate from fiber by gut microbiota are histone deacetylase inhibitors useful for controlling inflammation. Studies show that consumption of 105 mg sulforaphane contained in 68 g broccoli sprouts or 570 g mature broccoli can significantly inhibit histone deacetylase activity after 3 h. Sulforaphane has also been demonstrated to be useful in promoting detoxifying enzymes required to metabolize pesticides.
Despite these outcomes, additional epidemiological studies are required on the efficacy of organic food or supplements to contrast pesticide-induced damage, together with an evaluation of pesticide residue metabolites in biological fluids that provide data on human exposure and the individual genetic capacity to metabolize these chemicals.
References Bara nski, M., Srednicka-Tober, D., Volakakis, N., Seal, C., Sanderson, R., Stewart, G.B., Benbrook, C., Biavati, B., Markellou, E., Giotis, C., Gromadzka-Ostrowska, J., Rembiałkowska, E., Skwarło-So nta, K., Tahvonen, R., Janovska´, D., Niggli, U., Nicot, P., Leifert, C., 2014. Higher antioxidant and lower cadmium concentrations and lower incidence of pesticide residues in organically grown crops: a systematic literature review and meta-analyses. Br J Nutr 112 (5), 794e811. Collotta, M., Bertazzi, P.A., Bollati, V., 2013. Epigenetics and pesticides. Toxicology 307, 35e41. Declerck, K., Remy, S., Wohlfahrt-Veje, C., Main, K.M., Van Camp, G., Schoeters, G., Vanden Berghe, W., Andersen, H.R., 2017. Interaction between prenatal pesticide exposure and a common polymorphism in the PON1 gene on DNA methylation in genes associated with cardio-metabolic disease risk-an exploratory study. Clin Epigenetics 9, 35. Dennis, K.K., Jones, D.P., 2016. The exposome: a new frontier for education. Am Biol Teach 78, 542e548. Farina, Y., Abdullah, M.P., Bibi, N., Khalik, W.M., 2017. Determination of pesticide residues in leafy vegetables at parts per billion levels by a chemometric study using GC-ECD in Cameron Highlands, Malaysia. Food Chem 224, 55e61. Karmaus, A.L., Trautman, T.D., Krishan, M., Filer, D.L., Fix, L.A., 2017. Curation of food-relevant chemicals in ToxCast. Food Chem Toxicol 103, 174e182. Lampe, J.W., 2007. Diet, genetic polymorphisms, detoxification, and health risks. Altern Ther Health Med 13 (2), S108eS111. McKelvey, W., Jacobson, J.B., Kass, D., Barr, D.B., Davis, M., Calafat, A.M., Aldous, K.M., 2013. Population-based biomonitoring of exposure to organophosphate and pyrethroid pesticides in New York city. Environ Health Perspect 121 (11e12), 1349e1356. Skinner, M.K., 2016. Endocrine disruptors in 2015: epigenetic transgenerational inheritance. Nat Rev Endocrinol 12 (2), 68e70.
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