Pesticides exposure and premature thelarche: experimental and epidemiological tools for toxicological assessment

Pesticides, widely used in agriculture to protect plants and produce a high quality crop, cause serious human health concerns due to a high number of associated comorbidities such as cancer, neurological, cardiovascular and eproductive diseases including premature thelarche and early puberty. In addition, several pesticides are identified as endocrine disruptors because they may interfere with the functions of the hormonal system. Children due to their developmental stage, peculiar lifestyle and eating habits are more susceptible to the adverse effects of endocrine disruptors. The present study is within the framework of the project “Integrated approach to evaluate children agricultural pesticide exposure and health outcome” (PEACH project) and deals with two aspects converging in the toxicological risk assessment, i.e., the hazard identification and the exposure assessment. Specifically, the study is focused on: (a) the evaluation of the potential toxicological effects of three pesticides widely used in agriculture, such as chlorpyrifos (CPF), imidacloprid (IMI) and glyphosate (GLY), through an in vitro study comparing the response of two mammary cell lines; (b) the setting up of a questionnaire to be used within the epidemiological study of PEACH project, to design a sampling plan of food locally produced and consumed by the enrolled girls for pesticide residues determination and dietary exposure assessment. In vitro toxicological study The possible toxicological effects of CPF, IMI and GLY were evaluated by an in vitro study using two human mammary gland cell lines: MCF-7 a breast cancer cell line, commonly used, and MCF-12A a non-tumorigenic cell line, that, although barely used in toxicological studies, could be a more appropriate model simulating the "effective cellular condition" of healthy people; therefore 5 it may be more suitable to investigate the effects of pesticide exposure and the possible association with the idiopathic premature thelarche (IPT), as clue of pubertal disorders. In this respect, the ultimate goal of the study was to set up a three-dimensional (3D) cell culture system reproducing the in vivo physiology of the mammary gland, by using the selected cell lines as preparatory step for further studies. To evaluate the effects on cell viability and metabolism and endocrine activity, both cell lines were treated with the three pesticides at concentrations derived from real exposure values in children as reported by epidemiological data. The cytotoxic potential of the substances was measured by CyQUANT® and MTS assays able to detect cell proliferation and cell viability, respectively; cells were treated with five ten-fold serial dilutions of CPF (120 pM- 1.2 μM), IMI (160 pM–1.6 μM) and GLY (230 pM- 2.3 μM). In order to evaluate if the three pesticides might induce oxidative stress, altered the intracellular ATP production as well as apoptosis or necrosis, appropriate assays were performed in both cell line i.e., Reactive Oxygen Species (ROS) and Adenosine triphosphate (ATP) detection and Annexin V assays were performed in both cell lines. Furthermore, the endocrine activity of CPF, IMI and GLY was elucidated by the evaluation of their ability to interfere with estradiol production as well as the gene expression of specific nuclear receptors involved in the development of the mammary gland including estrogen receptors α and β (ERα ERβ), androgen receptor (AR), progesterone receptor (PgR) and Aryl hydrocarbon receptor (AhR). Specifically, estradiol concentration and gene expression were determined by Estradiol ELISA assay and real-time PCR, respectively, by using three ten-fold serial dilutions of CPF (1.2, 12 or 120 nM), IMI (1.6, 16 or 160 nM), GLY (2.3, 23 or 230 nM). Cell proliferation and cell viability were differently affected in MCF-7 and MCF-12A cell lines. In MCF-7 cells, only two pesticides exerted an effect on cell 6 proliferation at different concentrations: CPF at the highest concentration tested induced an increase while GLY at the lowest induced a decrease. In MCF-12A, all three pesticides induced a dose-dependent decrease in cell proliferation. The results showed that IMI and GLY are able to affect cell proliferation differently depending on the cell model postulating MCF-12A as a more sensitive model. The effects on cell proliferation in MCF-12A are supported by an increased apoptotic signal for GLY indicating activation of the apoptotic mechanism in the cell death process, whereas in MCF-7 cells no significant effect in apoptotic or necrotic signals was observed. In MCF-7 cells, all three pesticides induced a dose-dependent decrease in cell viability although in a different pattern, while in MCF-12A cells, CPF induced a dose-dependent decrease in cell viability, IMI at the lowest concentration tested induced an increase in cell viability and GLY had no effect. The decrease in cell viability observed in MCF-7 cells was supported by the decrease in ATP level induced by all three pesticides. No effect in ATP production was observed in MCF-12A. In MCF-7 cells, CPF at the two highest concentrations induced a decrease in ROS production, in contrast, IMI at the lowest concentration induced an increase in ROS production, whereas no effect was recorded for GLY. In MF-12A cells, intracellular ROS production was decreased by all three pesticides. In both cell lines, all three pesticides increased estradiol (E2) secretion although in different patterns. Furthermore, in MCF-7 cells, ERα gene expression was induced by CPF and IMI, whereas it was downregulated by GLY; all three pesticides downregulated ERβ and PgR gene expression and upregulated AR although with different patterns; moreover, IMI e GLY upregulated also AhR although with different pattern. 7 In MCF-12A cells, ERα and ERβ gene expression was upregulated by IMI and GLY; the same pesticides downregulated AR, PgR and AhR although with different pattern; only CPF downregulated ERβ with no effect on the other receptors. The final step of the in vitro study was to set up a 3D cell culturing that closely mimic the in vivo mammary gland. For this purpose, a hydrogel system and a magnetic system were set up and compared by using MCF-7 and MCF-12A cell lines, respectively. The magnetic system aggregating cells under magnetic forces appeared to be as a more suitable 3D cell culturing to study the effects of chemicals providing more reproducible experimental methodology and conditions. Epidemiological study: Food Frequency Questionnaire The aim was to develop a food frequency questionnaire to be filled in by the girls (or their parents) enrolled in the case-control study performed in the PEACH project. The food frequency questionnaire is divided into food groups (i.e., fruit, vegetables, meat, fish, cereals, eggs, oil) and food commodities. For each commodity, the quantity consumed, the frequency of consumption and the place of purchase are required. Data from the structured questionnaire were elaborated to sample food locally produced and consumed by girls, for pesticide residues determination. Food samples were collected in 17 local farms and 25 private gardens. At the end of PEACH project, data from questionnaires will be also used to estimate pesticide dietary intake. Overall, the results of this study, including both the in vitro toxicological study and the epidemiological study, provide evidence on pesticides toxicology and suitable tools for chemical risk assessment.