Currently used pesticides and their mixtures affect the function of sex hormone receptors and aromatase enzyme activity
Introduction
Exogenous compounds that alter the normal functioning of the endocrine system are called endocrine-disrupting chemicals (EDCs), referring to their potential to cause adverse health effects in humans and wildlife. Based on several in vitro and in vivo studies, a considerable number of environmental contaminants have been identified as EDCs and among them are several pesticides (Andersen et al., 2002, Andersen et al., 2008, Birkhoj et al., 2004, Grunfeld and Bonefeld-Jorgensen, 2004, Hofmeister and Bonefeld-Jorgensen, 2004, Long et al., 2003, Nellemann et al., 2003, Vinggaard et al., 2005a, Vinggaard et al., 2005b, Vinggaard et al., 2002).
The endocrine-disrupting (ED) potential of many pesticides is of health concern, because their residues are found not only in the environment but also in many food items as a result of the ubiquitous nature of pesticide usage with minimal precautions. Therefore, humans are exposed to pesticides via various routes throughout life, including dietary and environmental exposure (water, soil, air) and also through occupational settings. Several studies have shown the presence of pesticide residues and their metabolites in human tissues, demonstrating a global distribution (Arrebola et al., 2012, Jaga and Dharmani, 2003, Sanghi et al., 2003, Zietz et al., 2008).
In particular, the activity of estrogen-mimicking compounds has been under investigation for the last decades. Studies have demonstrated that a broad range of pesticides possess estrogenicity in vivo, e.g., methoxychlor, kepone and dichlorodiphenyltrichloroethane (DDT), and/or in vitro, e.g., o,p′-DDT, p,p′-DDT, endosulfan, toxaphene, dieldrin, methiocarb, prochloraz, fenarimol, triadimefon and triadimenol (Andersen et al., 2002, Bonefeld Jorgensen et al., 1997, Bonefeld-Jorgensen et al., 2005, Grunfeld and Bonefeld-Jorgensen, 2004, Soto et al., 1994). Estrogens are sex hormones of great importance for the female reproductive system. Moreover, they play key roles in both male and female fetal development and are essential for growth, differentiation and function of a broad range of tissues, e.g. the central nervous system, the musculoskeletal system, the immune system and the cardiovascular system (Bjornstrom and Sjoberg, 2005, Heldring et al., 2007). Thus, any disruption of estrogen receptor (ER) signaling pathways can contribute to adverse health effects such as infertility, developmental abnormalities and endocrine-related cancer (Bonefeld-Jorgensen et al., 2011, Mueller, 2004, Sikka and Wang, 2008).
Studies have shown that various pesticides, such as vinclozolin, dichlorodiphenyldichloroethylene (p,p′-DDE), prochloraz, procymidone, linuron, methoxychlor, dieldrin and fenitrothion, exert antiandrogenic effects by interfering with the androgen receptor (AR) (Andersen et al., 2002, Luccio-Camelo and Prins, 2011, Vinggaard et al., 2002). The AR is the key regulatory element of androgen cell signaling. Physiologically, AR-regulated gene expression is responsible for male sexual differentiation in utero and male reproductive function and development, including spermatogenesis (Dehm and Tindall, 2007, Gao et al., 2005).
Adverse trends in male reproductive health have appeared during the last decades (Andersson et al., 2008, Carlsen et al., 1992), and a synchronized increase in the incidence of male reproductive problems (e.g. testicular cancer, genital abnormalities, reduced semen quality and subfertility) has been hypothesized (Skakkebaek, 2003, Skakkebaek et al., 2001). Several studies have indicated a correlation between pesticide exposure and reduced male fertility (Lifeng et al., 2006, Oliva et al., 2001, Padungtod et al., 2000, Swan et al., 2003) and female fertility (Abell et al., 2000). Moreover, epidemiological studies have suggested a link between environmental exposure to pesticides and the risk of hormone-related cancer such as breast cancer and prostate cancer (Mnif et al., 2011, Sankpal et al., 2012).
There is a special concern regarding ED effects on growth and development of the embryo/fetus, being sensitive to hormonal fluctuations (Bigsby et al., 1999, Skakkebaek et al., 2001, Swan et al., 2005). Exposure to EDCs during early life stages is suspected to cause irreversible damage and developmental abnormalities, which may not be manifested until later in life. It is assumed that fetuses (i.e., pregnant women) and children are especially vulnerable groups concerning pesticide exposure. Studies have indicated an impaired reproductive development, including an increased prevalence of cryptorchidism, in sons of women working as gardeners or living on farms where pesticides have been used (Andersen et al., 2008, Carbone et al., 2007, Komarek et al., 2010, Kristensen et al., 1997, Weidner et al., 1998, Wohlfahrt-Veje et al., 2011).
So far, most pesticide research has been focusing on persistent organochlorine insecticides (e.g., DDT, chlordane and dieldrin) due to their former extensive use and bioaccumulative properties. However, during the last decades their use has been largely superseded by pesticides which do not persist appreciably in the environment. Therefore, it is imperative to further investigate the potential impact on human health of currently used “non-persistent” pesticides.
The present study was part of the cross-disciplinary research project HOPE, aiming for assessment of the ED potential of currently used pesticides in Denmark from different pesticide groups: phenoxy acids, triazines, sulfonylureas, growth regulators, azoles, dithiocarbamates, pyrethroids and organophosphates (Table 1). A dithiocarbamate metabolite was included in the study as well, because the mother compound, mancozeb, decomposes when exposed to moisture and air and has low persistence in soils (Komarek et al., 2010). Apart from the organophosphate insecticide malathion (banned in Denmark since 2008 but included in the study because of an extensive Danish fruit import) and the dithiocarbamate metabolite, the test compounds represent active ingredients in pesticide formulations commonly used in Denmark today.
The test compounds were selected on the basis of (i) application of pesticide, (ii) amount of pesticide (in kg) used in Denmark in 2006, (iii) acreage (in hectares) treated with pesticide in Denmark, and (iv) knowledge from published evidence of ED effects of pesticides and ongoing pesticide research (based on an extensive survey of EU assessment reports and additional literature search).
Herbicides such as the phenoxy acids, triazines and sulfonylureas are generally used to reduce weeds in various agricultural and horticultural settings. The phenoxy acids are post-emergence growth inhibitors applied to protect crops such as cereal grains, corn, fruit trees and some vegetables (Barr and Needham, 2002, Santilio et al., 2009). The triazines are broad-spectrum pre- and post-emergence herbicides used to control broadleaf and grassy weeds in various crops such as corn, sorghum, sugar cane etc. and similarly, sulfonylurea herbicides are used to control weeds in a variety of crops and vegetables (Barr and Needham, 2002, Sarmah and Sabadie, 2002). Plant growth regulators are important components in agricultural production as well. In grain crops, these compounds are used to reduce the length and strengthen the straw in order to prevent lodging and thus reduce the risk of difficulties during harvesting (Sorensen et al., 2009).
Various pesticides have been described for their activity against a broad range of fungi and insects. The azoles and dithiocarbamates are extensively used in plant protection worldwide due to their antimycotic properties. As the organochlorine pesticides were phased out in most countries, alternative insecticides such as the organophosphates and pyrethroids have become widely used to control insect pests in a variety of crops. Exposure to synthetic pyrethroids is expected to increase among the general population due to regulatory restrictions on the use of common organophosphates in the USA and other countries (Meeker et al., 2008).
The specific objectives of the present study were to evaluate in vitro the effect of the pesticides (Table 1) on ER and AR function as well as aromatase activity. The aromatase, encoded by the CYP19, is the key enzyme in the biosynthesis of estrogens from cholesterol, since it catalyzes the final rate-limiting step in which androgens are converted to estrogens. Consequently, the aromatase enzyme is crucial for the maintenance of the homeostatic balance between estrogen and androgen sex hormones. Aromatase activity (i.e., regulation of estrogen synthesis from androgens) is thought to be a critical endpoint concerning sexual development and differentiation (Jones et al., 2006).
As humans are continuously exposed to multiple EDCs simultaneously, a growing concern regarding the risk of mixture effects has emerged. Several in vitro and in vivo studies have demonstrated mixture effects of pesticides (Birkhoj et al., 2004, Hass et al., 2012, Jacobsen et al., 2012, Kjaerstad et al., 2010a, Nellemann et al., 2003) and other EDCs such as plasticizers and phenols (Ghisari and Bonefeld-Jorgensen, 2009, Kruger et al., 2008). In addition to the single pesticides, we also analyzed two mixtures selected on the basis of observed in vitro and in silico potencies of the single test compounds in the HOPE project, including analyses of hormone receptor interference, aromatase enzyme activity, steroid hormone synthesis and quantitative structure–activity relationship (QSAR).
Section snippets
Chemicals
The high-affinity ER ligand 17β-estradiol (E2) was obtained from Sigma-Aldrich (Denmark) and used as positive dose–response control in the ER transactivation assay. E2 was dissolved in 96% extra pure ethanol from Merck (Darmstadt, Germany) to produce a 100 nM stock solution.
The AR agonists, methyltrienolone (R1881) and dihydrotestosterone (DHT) were obtained from Perkin Elmer (Hvidovre, Denmark) and Sigma-Aldrich (Denmark), respectively. The AR antagonist hydroxyflutamide (HF) was from MikroMol
Cytotoxicity
Effects of the pesticides on ER transactivation were analyzed in MVLN cells. Bitertanol, prothioconazole and cypermethrin displayed a cytotoxic response at concentrations ≥ 5 × 10− 5 M, whereas propiconazole and mancozeb were toxic to the MVLN cells at concentrations ≥ 1 × 10− 4 M. In addition, an incipient toxicity at 1 × 10− 4 M was observed for 2-methyl-4-chlorophenoxyacetic acid (MCPA), iodosulfuron-methyl-sodium, metsulfuron-methyl, chlormequat chloride, tau fluvalinate and malathion. The pesticide
Discussion
The present study was undertaken to evaluate in vitro the effects of 13 pesticides and one metabolite on ER and AR transactivity and aromatase enzyme activity. Our data demonstrate the potential of five pesticides (terbuthylazine, propiconazole, prothioconazole, cypermethrin and malathion) to weakly induce the ER transactivity, and three pesticides (bitertanol, propiconazole and mancozeb) to cause concentration-dependent antiandrogenic effects. Moreover, three pesticides (terbuthylazine,
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
We thank the members of the Centre for Arctic Health research group, especially Tanja Krüger for the technical and scientific support regarding the AR transactivation assay and Dorte Olsson for the technical assistance. The present study was part of the research project HOPE (Hormone Disrupting Effect of Currently used Pesticides) aiming for assessment of the ED potential of currently used pesticides in Denmark from different pesticide groups. We thank the HOPE partner team for the scientific
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