Open access peer-reviewed chapter

The Pragmatic Strategy to Detect Endocrine-Disrupting Activity of Xenobiotics in Food

Written By

Shui-Yuan Lu, Pinpin Lin, Wei-Ren Tsai and Chen-Yi Weng

Submitted: 30 May 2018 Reviewed: 20 August 2018 Published: 05 November 2018

DOI: 10.5772/intechopen.81030

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Medicinal Chemistry

Edited by Janka Vašková and Ladislav Vaško

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Abstract

Endocrine-disrupting activity induced by xenobiotics might pose a possible health threat. Facing so many chemicals, there is an issue on how we detect them precisely and effectively. The whole embryo culture (WEC) test, an ex vivo exposure lasting 48 hours with rat embryos of 10.5 days old, is used to detect prenatal developmental toxicity. We extended the WEC function to detect the endocrine-disrupting activity induced by environmental chemicals. Results showed that in the development of rat embryo, basically 17ß-estradiol, triiodothyronine, triadimefon, penconazole, and propiconazole exhibited no significant effect on yolk sac circulatory system, allantois, flexion, heart caudal neural tube, hindbrain, midbrain, forebrain, otic system, optic system, olfactory system, maxillary process, forelimb, hind limb, yolk sac diameter, crown-rump length, head length, and developmental score. In the immunohistochemistry, the positive control of 17ß-estradiol showed positive effect for its receptor expressions. These three triazoles induced expressions of ERα and ERß in WEC. This result basically meets the mode of action that triazoles were designed to disrupt the synthesis of steroid hormone. Here we gave a strategy to detect possible endocrine-disrupting activity induced by xenobiotics in food. This strategy is quick to initiate the whole rat embryo culture with 10.5 days to detect the hormone receptors such as androgen, estrogen, thyroid, aromatase activity and its related receptors.

Keywords

  • whole embryo culture
  • xenobiotic
  • receptors
  • ex vivo
  • in vivo
  • endocrine-disrupting activity

1. Introduction

As we know, there are many pesticides identified as endocrine disruptors, but the degree of endocrine-disrupting activity (EDA) is different [1, 2, 3, 4, 5]. The different disrupting activities are involved in pesticide management. Because the potential endocrine-disrupting pesticides should be prohibited, low EDA will be accepted under the control of below maximum residue level (MRL). The development of new pesticide is based on its chemical functional groups for pests including fungicides, insecticides, herbicides, and others. Due to the objective of pest control of diseases, insects, and weeds, the side effect of pesticides will be appropriately managed in order not to pose risk to the human and environment. It is reported that 105 pesticides could be listed in the endocrine-disrupting chemical (EDC) group (Table 1) [6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54]. Among these 105 pesticides, 31% are fungicides, 21% herbicides, and 46% insecticides; some of these were withdrawn from use several years ago; even a little still can be detected in the environment such as dichloro-diphenyl-trichloroethane (DDT) and atrazine in some countries.

Pesticides EDC related Pesticides EDC related
2,4-D (H) AR [6] Heptachlor (I) ER, AR [25, 46]
Acephate (I) Hypothalamus [7] Hexaconazole (F) Aromatase activity, estrogens, androgens [20]
Acetochlor (H) ER, TR [8, 9] Isoproturon (H) Pregnane X cellular receptor [5]
Alachlor (H) ER, PR [10, 11] Iprodione (F) Aromatase activity, estrogen [2]
Aldicarb (I) 17 Beta-estradiol, progesterone [10, 12] Linuron (H) AR, TR [25, 47]
Aldrin (I) AR [13] Malathion (I) TR [10, 48]
Atrazine (H) Androgen, aromatase activity, estrogen, luteinizing hormone, prolactin [10, 14, 15, 16, 17] Methiocarb (H) Androgen, estrogen [2]
Bendiocarb (I) Estrogen effect [10] Methomyl (I) Aromatase activity, estrogen [2, 10]
Benomyl (F) Estrogen, aromatase activity [18] Methoxychlor (I) Estrogenic effect, AR, pregnane X cellular receptor [10, 11, 13]
Bioallethrin (I) Estrogen-sensitive [19] Metolachlor Pregnane X cellular receptor [5]
Bitertanol (F) Aromatase activity, estrogens, androgen [20] Metribuzin (H) Hyperthyroidism, somatotropin [49]
Bupirimate (F) Pregnane X cellular receptor [5] Mirex (I) Estrogen effect [10]
Captan (F) Estrogen action [21] Molinate (H) Reduction of fertility [10]
Carbaryl (I) Estrogen effect [10] Myclobutanil (F) Estrogen, androgen, ER, AR, aromatase [20, 21, 35]
Carbendazim (F) Estrogen and aromatase activity [18] Nitrofen (H) Estrogen, androgen [21]
Carbofuran (I) Progesterone, cortisol, estradiol, testosterone [22] Oxamyl (I) Estrogen effect [10]
Chlorothalonil (F) Androgen-sensitive [23] Parathion (I) Melatonin, gonadotrophic hormone [10]
Chlordane (I) ER [10], AR [13] Penconazole (F) Estrogenic effect, aromatase activity, estrogens, androgens [20, 35]
Chlordecone (I) AE, ER [21, 24, 25] Pentachlorophenol (H, F, I) Estrogenic, androgenic affect [10]
Chlorfenvinphos (I) Estrogen effect [26] Permethrin (I) Estrogen-sensitive [19, 29]
Chlorpyrifos methyl (I) AR [27] Phenylphenol (F) Estrogen [50]
Cypermethrin (I) Estrogenic effect [28, 29] Prochloraz (F) Pregnane X cellular receptor, AR, ER, AhR, aromatase activity [2, 5, 36, 51]
Cyproconazole (F) Aromatase activity, estrogens, androgens [20] Procymidone (F) AR [25]
DDT and metabolites (I) AR, androgen-sensitive, ER, PR [13, 23, 24, 30] Propamocarb (F) Aromatase activity, estrogen [2]
Deltamethrin (I) Estrogenic activity [2] Propanil (H) Estrogen [52]
Diazinon (I) Estrogenic effect [31] Propazine (H) Aromatase activity, estrogen [15]
Dichlorvos (I) AR [2] Propiconazole (F) Estrogen, aromatase activity, androgens [20, 35]
Dicofol (I) Androgen synthesis, estrogens synthesis, ER [17, 21] Propoxur (I) Estrogenic effect [10]
Dieldrin (I) AR, estrogenic effect, ER [2, 13, 24, 32] Prothiophos (I) Estrogenic effect [31]
Diflubenzuron (I) Pregnane X cellular receptor [5] Pyridate (H) ER, AR [21]
Dimethoate (I) Thyroid hormones, insulin, luteinizing hormone [33, 34] Pyrifenox (F) Estrogen [35]
Diuron (H) Androgen action [17] Pyriproxyfen (I) Estrogenic effect [31]
Endosulfan (I) AR, estrogenic effect, ER, aromatase activity [2, 13, 30, 32] Resmethrin (I) Sex hormone [40]
Endrin (I) AR [13] Simazine (H) Aromatase activity, estrogen [15]
Epoxiconazole (F) Aromatase activity, estrogen, androgens [20, 35] Sumithrin (I) Estrogen-sensitive, progesterone [19, 39]
Fenarimol (F) Androgenic action, aromatase, pregnane X cellular receptor [2, 5, 36] Tebuconazole (F) Aromatase activity, estrogens, androgens [20]
Fenbuconazole (F) Thyroid hormones, pregnane X cellular receptor [5, 10] Tetramethrin (I) Estrogen [53]
Fenitrothion (I) AR, estrogens [21, 37] Tolclofos-methyl (I) ER [36]
Fenoxycarb (I) Testosterone [38] Toxaphene (I) Estrogen-sensitive, corticosterone [10, 32]
Fenvalerate (I) Estrogen-sensitive, progesterone [18, 39] Triadimefon (F) Estrogenic effect, aromatase activity, androgens [21]
Fluvalinate (I) Human sex hormone, progesterone [40, 41] Triadimenol (F) Estrogenic effect, aromatase activity, androgens [20, 21]
Flusilazole (F) Aromatase activity, estrogens, androgens [20] Tribenuron-methyl (H) Estrogenic effect [2]
Flutriafol (F) Estrogen [35] Trichlorfon (I) Thyroid function [54]
Glyphosate (H) Aromatase activity, estrogens [42] Trifluralin (H) Pregnane X cellular receptor, steroid hormone [11]
HCB (F) Thyroid hormone, androgen [43, 44] Vinclozolin (F) AR, pregnane X cellular receptor, steroid hormone [2, 11, 25]
HCH (lindane) (I) Estrous cycles, luteal progesterone, insulin, estradiol, thyroxine, AR, ER, PR [33, 45]

Table 1.

The summary of reported endocrine disruptor pesticides and their related EDC activity.

I, insecticides; F, fungicides; H, herbicides

EDCs focused on interfering with endogenous hormones possible by binding to and activating various hormone receptors including estrogen, androgen, thyroid receptors, and aromatase enzymes and mimic the hormone or enzyme activities including agonistic and antagonistic actions. Basically, EDA is mainly related to the reproductive and developmental toxicity. Also the major endocrine pathways would be hypothalamus-pituitary-gonadal and hypothalamus-pituitary-thyroid, and the involving hormones are estrogen, androgen, and thyroid. The Organization for Co-operation and Development (OECD) test guidelines for reproductive and developmental toxicity and EDA are listed in Table 2 [55, 56]. United States Environmental Protection Agency (US EPA) test guidelines for reproductive and developmental toxicity and EDA are as follows. Guidelines are 870.3550 reproduction/development toxicity screening test, 870.3650 combined repeated dose toxicity with the reproduction/development toxicity screening test, 870.3700 prenatal developmental toxicity study, 870.3800 reproduction and fertility effects, and 870.6300 developmental neurotoxicity study. USEPA Series 890 endocrine disruptor screening program test guidelines are isolated from OPPTS 870 Series. The final endocrine disruptor screening program test guidelines are generally intended to meet testing requirements under Toxic Substances Control Act (TSCA); Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA); and Federal Food, Drug, and Cosmetic Act (FFDCA) to determine if a chemical substance may pose a risk to human health or the environment due to the disruption of the endocrine system. Group A—EDSP Tier 1 and Group B—EDSP Tier 2 test guidelines are listed in Table 3.

OECD guideline Topic Animals Estimated cost (€)
414 Prenatal development toxicity 784 63,100 (rats)
92,500 (rabbits)
416 Reproductive toxicity in two generations 3200a 328,00
421 Screening test for reproductive and developmental toxicity 560 54,600
422 Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test 412 92,000
426 Neurodevelopmental toxicity study 1400 1100

Table 2.

Economical cost and number of animals needed to apply the OECD guidelines for testing reproductive toxicology.

All the animals including discarded pups


Data came from Rovida and Hartung [55]; Sogorb et al. [56].

OPPTS 890 series Topic
Group A—EDSP Tier 1
890.1100 Amphibian metamorphosis (frog)
890.1150 Androgen receptor binding (rat prostate)
890.1200 Aromatase (human recombinant)
890.1250 Estrogen receptor binding
890.1300 Estrogen receptor transcriptional activation (human cell line HeLa-9903)
890.1350 Fish short-term reproduction
890.1400 Hershberger (rat)
890.1450 Female pubertal (rat)
890.1500 Male pubertal (rat)
890.1550 Steroidogenesis (human cell line—H295R)
890.1600 Uterotrophic (rat)
Group B—EDSP Tier 2
890.2100 Avian two-generation toxicity test in the Japanese quail
890.2200 Medaka-extended one-generation reproduction test
890.2300 Larval amphibian growth and development assay (LAGDA)

Table 3.

USEPA Tier 1 and Tier 2 test guidelines.

The main shortcomings of above guidelines are that they are expensive and time-consuming and the need of a lot of number of laboratory animals. It is reported that cost and the minimum number of laboratory animals are requested for applying OECD test guidelines to test toxicity to reproductive and developmental toxicity. Table 2 shows the cost and minimum number of laboratory animals [55, 56]. Besides, the associated bioethical and social concerns are becoming a challenge. Nowadays, the common knowledge of using laboratory animals is reduce, refine, and replace (3Rs). Facing these situations, we should take cheap and reliable alternatives to screen the reproductive and developmental toxicity and EDA and decide the next steps for necessities of toxicity tests.

It is reported that a widely used technique for screening prenatal developmental toxicity is by monitoring organogenesis during gestational days (GD) 10–12 [57]. In support to whole rat embryo culture (rat WEC), a variety of morphological endpoints is integrated in the total morphological score (TMS) [58]. When applying the TMS in rat WEC, effects of pesticides on the embryonic toxicity could be investigated with qualitative and quantitative endpoints. As we know, azoles are antifungal agents for clinical and agricultural use. Penconazole, propiconazole, and triadimefon were most common triazole pesticides in Taiwan. A report showed that triazole chemicals antagonized the aromatase, which transfer testosterone into 17ß-estradiol in mammals. Triazole chemicals were designed to disrupt the Cyp51 enzyme, which catalyzes the conversion of lanosterol to ergosterol on the fungal cell membrane, and led to cell death when attacked [59]. Though in the respect of mammalian systems Cyp51 is less sensitive to azoles, it was still critical for the sterol biosynthesis pathway and might be related to the thyroid function. In this study, we will take triazoles penconazole, propiconazole, and triadimefon as an example for the alternative of endocrine-disruptor screening.

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2. Materials and methods

2.1. Animals

The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Taiwan Agricultural Chemicals and Toxic Substances Research Institute. Five-week-old male and female Wistar rats were purchased from BioLASCO (Taipei, Taiwan, ROC). The rats were acclimated to the laboratory environment and reared under a controlled temperature (21 ± 2 °C), humidity (40–70%), frequency of ventilation (at least 10/h), and alternating 12 hour cycles of light and darkness. The rats were administered a pellet rodent diet and water ad libitum until they were sacrificed. At 12 weeks of age, the 4 male and 20 female rats were allowed to mate with 2 males to 2 females per day. Gestation day (GD) 0 was defined as the day that sperm was observed in the vagina of the female following mating.

2.2. Chemicals

Materials were obtained from the following manufacturers: DMSO (dimethyl sulfoxide), T3 (triiodothyroxine), Tria (triadimefon), Penc (penconazole), and Prop (propiconazole). All these chemicals with 97% pure at least were purchased from Sigma Chemical Co. (St. Louis, MO).

2.3. Rat whole embryo culture

Five-week-old female and male rats were purchased and reared in the first animal house breeding room until 11–12 weeks of age. Two males and two females were bred in the same cage. The female rats were examined for vaginal plugs on the next day. The occurrence was considered as successful breeding. From the date of pregnancy to the 10.5th day, the embryos were dissected. Reichert’s membrane was removed according to the method described by Andrews et al. [60] and Dimopoulou et al. [61], and the embryos containing the intact yolk sac placenta and the urinary membrane were removed and randomly placed in a 4 mL culture medium HBSS solution containing 50 IU of penicillin G/mL and 50 μg streptomycin/mL. The sample was added to a 25 T culture flask containing filter-sterilized rat serum and subjected to complement deactivation and cultured in a constant temperature incubator at 37°C for 48 hours. The culture solution was initially inflated with a mixed gas of 5% O2, 5% CO2, and 90% N2 for 1 minute, and after about 16 hours of culture, 10% O2, 5% CO2, and 85% N2, inflated for 1 minute, and were cultured until the 24th hour. Inflate for 1 minute with 20% O2, 5% CO2, and 75% N2. Each treatment dose was inflated for 1 minute at 40% O2, 5% CO2, and 55% N2 at 40 hours, and the embryos were measured for growth, development, and morphology at the end of 48 hours of culture. Embryonic development was modified according to Brown and Fabro [62], and the evaluation included embryo growth traits and developmental stages, which were considered death if the embryonic yolk sac circulation system or the heart stopped beating. Finally, the carcass head-tail length, developmental grade, head length, number of body segments, and yolk sac diameter were analyzed by t-test and related measurements according to statistical methods; death and abnormal embryos were determined by chi-square. Half of the evaluated embryos were preserved in neutral formalin solution for immunostaining, and the other half were stored in PBS for WB analysis to detect antibody responses related to hormone receptor or enzyme antibodies including AR, ERα, ERß, TRα, TRß, and aromatase.

2.4. Pesticide treatment and evaluation of embryo morphology

This study aimed to investigate the effect of these three pesticides on estrogen receptor (ERα and ERß), thyroid receptor (TRα and TRß), and aromatase activities in whole rat embryo culture (rat WEC) on gestation day (GD) 10.5. The concentrations of WEC were 3.1E-5, 6.2E-5, and 1.2E-4 M of penconazole, propiconazole, and triadimefon. The culture period was 48 hours. After culture the embryo morphology was assessed according to the TMS system [62], we graded the endpoint as no effect (−), little effect (±), effect (+), and potential effect (++). After evaluation of embryo development, it was fixed in formalin or kept in HBSS for immunohistochemistry (IHC) and western blot (WB), respectively.

2.5. Immunohistochemical (IHC) evaluation

The embryos were treated by penconazole, propiconazole, and triadimefon with concentrations of 3.1E-5, 6.2E-5, and 1.2E-4 M. Embryos from control and pesticide treatments were fixed in 10% neutral buffered formalin for 1 week. The embryos were then dehydrated with increasing concentrations of ethanol, cleared in toluene, and embedded in paraffin. All the sections were cut into 5 mm slices and deparaffinized, hydrated, and treated with 0.3% H2O2 in PBS (pH 7.6) for 30 minutes to block endogenous peroxidase activity and finally treated with a protein-blocking solution (5% goat serum diluted in phosphate-buffered saline). All these steps were followed by heating the sections in a microwave oven for antigen retrieval using a 0.01 M citrate buffer solution (pH 5.5). Tissue sections were immunostained with rabbit anti-AR(N-20), anti-ER (MC) antibody (Santa Cruz Co., CA), TRα (C0345), TRß (C0346) (Assay Biotechnology Co. Sunnyvale, CA), and aromatase (SM2222P)(Acris Antibodies, Inc., San Diego, CA), which was diluted 1:250 in phosphate-buffered saline and 0.25% bovine serum albumin and maintained at room temperature overnight. The tissue sections were then developed with a streptavidin-HRP kit (Chemicon IHC Select® CA, USA), using diaminobenzidine as the chromogen, and were counterstained with hematoxylin. All images were optimized by using an inverted microscope (Leica, Wetzlar GmbH, Germany). To quantify the relative amount of activity of ER, TR, and aromatase in the IHC, 200 nuclei stained per field in a slide, 5 fields per slide, and 5 slides per dose were counted. The intensity of AR, ER, TR, and aromatase proteins stained in nucleus was graded as (0, negative), + (1, mild), ++ (2, moderate), +++ (3, intense), ++++ (4, more intense), or +++++ (5, very intense). The measurements were control group adjusted, and the values were statistically analyzed.

2.6. Western blot

The embryo homogenates were then centrifuged at 3000 × g for 30 minutes at 4°C. The supernatants were aliquoted and stored at −86°C before use. Before western blotting, protein contents were measured by BCA protein assay (Cat. No. 23225, Pierce). Equal amounts of protein were loaded onto each polyacrylamide gel. The antibody dilutions were 1:200 for the anti-AR (N-20), ERα (MC-20), ERß (H-150) (Santa Cruz Co., CA), TRα (C0345), TRß (C0346) (Assay Biotechnology Co. Sunnyvale, CA), and aromatase (SM2222P) (Acris Antibodies, Inc., San Diego, CA) and 1:5000 for the horseradish peroxidase-conjugated goat anti-rabbit IgG (AP132P, Chemicon International). For each treatment group, five samples were analyzed in two separate blots. Total protein extracts from the embryo homogenates were denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 7.5% polyacrylamide. The proteins were transferred to nitrocellulose membranes. The membranes were then blocked for non-specific binding and incubated with polyclonal primary antibodies for AR (N-20), ERα (MC-20), ERß (H-150) (Santa Cruz Co., CA), TRα (C0345), TRß (C0346) (Assay Biotechnology Co. Sunnyvale, CA), aromatase (SM2222P)(Acris Antibodies, Inc., San Diego, CA), and ß-actin (AP132P, Chemicon International). After incubation with primary antibody, the membranes were incubated with horseradish peroxidase-linked anti-goat IgG secondary antibody and visualized on film exposed to enhanced chemiluminescence (VisualizerTM Western Blot Detection Kit, Millipore, MA, USA). The relative amount of protein in the resulting immunoblot bands was estimated by measuring the optical densities of the bands on exposed films using a FOTO/Analyst® Investigator System (Fotodyne Incorporated, WI, USA). The measurements were background adjusted, and the values were statistically analyzed. Protein for ß-actin served as an internal standard.

2.7. Statistical analysis

The values of ER, TR, and aromatase in western blot were normalized against ß-actin. All results were statistically analyzed with the t-test, and p < 0.05 was considered statistically significant. The other data were expressed as mean ± SE. Data were subjected to ANOVA followed by t-test. The level of significance was set at p < 0.05.

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3. Results

In the development of rat embryo, 17ß-estradiol (E2), triiodothyronine (T3), triadimefon, penconazole, and propiconazole exhibited no significant effect on yolk sac circulatory system, allantois, flexion, heart caudal neural tube, hindbrain, midbrain, forebrain, otic system, optic system, olfactory system, maxillary process, forelimb, hind limb, yolk sac diameter, crown-rump length, head length, and developmental score (Tables 46; Figure 1).

Treatment Yolk sac circulatory system Allantois Flexion Heart Caudal neural tube Hindbrain Midbrain
DMSO 3.1 ± 0.3 4.0 ± 0.0 2.0 ± 0.8 2.6 ± 0.7 3.7 ± 1.3 2.8 ± 0.8 2.8 ± 0.8
E2 3.0 ± 0.8 4.0 ± 0.0 3.0 ± 1.8 2.8 ± 0.5 4.3 ± 1.0 2.0 ± 1.2 2.5 ± 1.0
T3 3.0 ± 0.0 4.0 ± 0.0 2.5 ± 2.1 2.0 ± 1.4 3.0 ± 0.0 3.0 ± 0.0 3.0 ± 0.0
Triadimefon
L 2.5 ± 0.6 4.0 ± 0.0 3.0 ± 0.8 3.0 ± 0.0 4.0 ± 0.0 1.0 ± 0.0 3.0 ± 0.0
M 2.8 ± 0.5 4.0 ± 0.0 2.5 ± 0.6 3.0 ± 0.0 4.3 ± 0.5 2.5 ± 1.0 2.5 ± 1.0
H 3.0 ± 0.0 4.0 ± 0.0 1.8 ± 1.0 2.8 ± 0.4 4.0 ± 0.9 2.7 ± 0.8 2.7 ± 1.0
Penconazole
L 3.7 ± 0.6* 4.0 ± 0.0 3.7 ± 1.2* 3.0 ± 0.0 4.3 ± 1.2 3.0 ± 0.0 3.0 ± 0.0
M 3.6 ± 0.6* 4.0 ± 0.0 2.7 ± 0.6 3.0 ± 0.0 4.0 ± 0.0 3.0 ± 0.0 3.0 ± 0.0
H 3.4 ± 0.5 4.0 ± 0.0 3.0 ± 1.9 2.4 ± 0.9 3.8 ± 0.8 2.2 ± 1.1 2.4 ± 0.9
Propiconazole
L 3.0 ± 0.0 4.0 ± 0.0 3.0 ± 2.0 3.0 ± 0.0 3.7 ± 0.6 2.3 ± 1.2 2.3 ± 1.2
M 2.8 ± 0.4 3.8 ± 0.4 2.4 ± 0.9 3.0 ± 0.0 4.0 ± 0.7 2.8 ± 0.4 2.8 ± 0.5
H 3.0 ± 0.8 4.0 ± 0.0 2.5 ± 1.7 3.0 ± 0.0 3.8 ± 1.0 3.0 ± 0.0 3.0 ± 0.0

Table 4.

Effect of treatment with triazole pesticides on some developmental scores of rat embryo culture of day 10.5 for 48 hours.

P < 0.05.


All pesticide concentrate are 3.1E-5 M (low concentration, L), 6.2E-5 M (middle concentration, M), and 1.2E-4 M (high concentration, H). Dimethyl sulfoxide, DMSO; 17ß-estradiol, E2; and triiodothyronine, T3. E2 and T3 concentrations, 1.2E-4 M.

Treatment Forebrain Otic system Optic system Olfactory system Branchial bars Maxillary process Mandibular process
DMSO 2.7 ± 0.7 1.8 ± 0.4 2.8 ± 1.3 1.5 ± 0.7 1.4 ± 0.5 0.9 ± 0.3 2.0 ± 0.0
E2 2.8 ± 1.3 2.0 ± 0.8 3.0 ± 1.4 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 2.0 ± 0.0
T3 3.0 ± 0.0 1.5 ± 0.7 2.5 ± 2.1 1.5 ± 0.7 1.5 ± 0.7 1.0 ± 0.0 2.0 ± 0.0
Triadimefon
L 2.8 ± 0.6 1.5 ± 0.6 3.5 ± 1.0 1.8 ± 0.5 1.3 ± 0.5 1.0 ± 0.0 2.0 ± 0.0
M 2.3 ± 1.0 1.5 ± 0.6 3.3 ± 1.0 1.0 ± 0.0 1.5 ± 0.6 1.0 ± 0.0 2.0 ± 0.0
H 2.7 ± 1.0 1.7 ± 0.5 3.3 ± 0.8 1.5 ± 0.5 1.2 ± 0.4 1.0 ± 0.0 2.0 ± 0.0
Penconazole
L 3.3 ± 0.6 1.7 ± 0.6 3.3 ± 1.2 1.7 ± 0.6 1.3 ± 0.6 1.0 ± 0.0 2.0 ± 0.0
M 3.7 ± 0.6 1.7 ± 0.6 4.0 ± 0.0 1.7 ± 0.6 1.7 ± 0.6 1.0 ± 0.0 2.0 ± 0.0
H 2.6 ± 1.5 2.0 ± 1.0 3.2 ± 1.8 1.2 ± 0.8 1.2 ± 0.4 1.0 ± 0.0 2.0 ± 0.0
Propiconazole
L 2.7 ± 1.5 1.7 ± 1.2 3.3 ± 2.1 1.3 ± 0.6 1.3 ± 0.6 1.0 ± 0.0 2.0 ± 0.0
M 2.8 ± 0.4 1.4 ± 0.5 2.6 ± 1.1 1.2 ± 0.4 1.2 ± 0.4 1.0 ± 0.0 2.0 ± 0.0
H 3.3 ± 0.5 1.5 ± 0.6 2.5 ± 1.7 1.8 ± 0.5 1.0 ± 0.0 1.0 ± 0.0 2.0 ± 0.0

Table 5.

Effect of treatment with triazole pesticides on some other developmental scores of rat embryo culture of day 10.5 for 48 hours.

All pesticide concentrate are 3.1E-5 M (low concentration, L), 6.2E-5 M (middle concentration, M), and 1.2E-4 M (high concentration, H). Dimethyl sulfoxide, DMSO; 17ß-estradiol, E2; and triiodothyronine, T3. E2 and T3 concentrations: 1.2E-4 M.

Treatment Forelimb Hind limb Yolk sac diameter (A) (mm) Yolk sac diameter (B) (mm) Crown-rump length (mm) Head length (mm) Developmental score
DMSO 0.7 ± 0.5 0.7 ± 0.5 6.4 ± 1.2 5.7 ± 1.0 5.2 ± 1.1 1.9 ± 0.6 38 ± 7
E2 0.8 ± 0.5 0.8 ± 0.5 6.6 ± 1.4 5.2 ± 1.7 4.4 ± 1.4 2.2 ± 0.7 38 ± 8
T3 1.0 ± 0.0 1.0 ± 0.0 5.8 ± 0.1 4.9 ± 1.6 4.0 ± 1.8 1.7 ± 0.8 38 ± 6
Triadimefon
L 0.5 ± 0.6 0.8 ± 0.5 4.8 ± 1.0 4.8 ± 0.6 4.9 ± 0.4 1.7 ± 0.3 40 ± 3
M 0.5 ± 0.6 1.0 ± 0.0 5.0 ± 0.7 5.0 ± 0.7 5.4 ± 1.0 1.9 ± 0.3 38 ± 2
H 0.7 ± 0.5 0.8 ± 0.4 4.7 ± 0.9* 5.3 ± 1.2 4.9 ± 0.9 1.8 ± 0.5 39 ± 4
Penconazole
L 0.7 ± 0.6 1.3 ± 0.6 7.1 ± 1.7 6.4 ± 1.6 6.0 ± 1.0 2.5 ± 0.6 43 ± 4
M 1.0 ± 0.0 0.7 ± 0.6 6.9 ± 0.5 5.7 ± 1.1 5.8 ± 0.7 3.0 ± 0.5 43 ± 3
H 0.6 ± 0.5 1.0 ± 0.7 6.3 ± 1.2 6.2 ± 1.2 4.6 ± 1.7 2.1 ± 1.1 39 ± 9
Propiconazole
L 0.7 ± 0.6 0.7 ± 0.6 6.0 ± 1.4 5.4 ± 0.8 5.0 ± 0.4 2.1 ± 0.7 38 ± 9
M 0.6 ± 0.5 0.6 ± 0.5 4.2 ± 0.8* 4.5 ± 1.0* 4.5 ± 1.1 1.8 ± 0.4 40 ± 5
H 0.7 ± 0.6 0.8 ± 0.5 5.5 ± 2.2 4.8 ± 0.5 4.2 ± 1.4 1.9 ± 0.8 38 ± 6

Table 6.

Effect of co-treatment with triazole pesticides on developmental parameters and scores of rat embryo culture of day 10.5 for 48 hours.

P < 0.05.


All pesticide concentrate are 3.1E-5 M (low concentration, L), 6.2E-5 M (middle concentration, M), and 1.2E-4 M (high concentration, H). Dimethyl sulfoxide, DMSO; 17ß-estradiol, E2; and triiodothyronine, T3. E2 and T3 concentrations: 1.2E-4 M.

Figure 1.

The rat whole embryo culture.

In the immunohistochemistry (IHC), the 17ß-estradiol (ERα and ERß) positive control showed the respective results of receptor expressions. Our results showed that penconazole, propiconazole, and triadimefon induced expressions of ERα (Figure 2) and ERß (Figure 3) in WEC. This result basically meets the mechanisms of triazoles designed to disrupt the synthesis of steroid hormone. Also, results showed that penconazole, propiconazole, and triadimefon induced expressions of TRß (data not shown), but not in TRα (data not shown) with WEC. The relationship among TRß and AR and ER still needs to be investigated. Also, we need to study the antagonistic effects by adding the antagonists for the receptor expression. These three pesticides did not affect significantly AR (data not shown) and aromatase activity (data not shown). In the western blot (WB) data, these three pesticides did not affect significantly AR, ERα, ERß, TRα, TRß, and aromatase expressions in WEC (data not shown). The difference between IHC and WB induced by these three pesticides might be the sensitivity of detecting method. WB needs some embryos for the protein quantitative, while IHC can detect activity in an embryo.

Figure 2.

Effect of penconazole, propiconazole, and triadimefon on ERalpha activity in WEC.

Figure 3.

Effect of penconazole, propiconazole, and triadimefon on ERbeta activity in WEC.

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4. Discussion

WEC was used to study the prenatal developmental toxicity induced by environmental chemicals including phthalate and methoxyacetic acid [63, 64], aliphatic amides [65], and triazole pesticides [66, 67]. In respect of the 3Rs principle of animal study, WEC is an alternative to screen the potential of prenatal developmental toxicity of environmental compounds. Although ex vivo exposure of WEC was used limitedly without metabolisms of chemicals, most chemicals exhibited their action by parent compound. In this study, we found that in combination with IHC and WB, WEC will be a robust way to detect the endocrine-disrupting activity induced by environmental chemicals. In this study, we used WEC to detect the important receptors including AR, ERα, ERß, TRα, and TRß and enzyme aromatase activity potential induced by triadimefon, penconazole, and propiconazole. There is one shortcoming of WEC to be addressed. Due to the small amount of embryo, WB is hard to quantify the proteins of hormone receptors. The solution to the problem is to pool the embryo treated by one dose and analyze it. Also, we knew that fortunately nowadays IHC quantification is available. Finally, we concluded that in combination with IHC and WB, WEC will be a robust way to detect EDCs in food.

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5. Future work and recommendations

In order to meet the 3Rs including reduction, refine, and replace and precise risk assessment, adverse outcome pathway (AOP) is extensively developed by OECD. By tier screening for EDCs, the molecular initiating event (MIE), key event (KE), key event relationship (KER), and adverse outcome (AO) will be studied. As the guideline stated, the AOP framework made clear the mechanisms from MIE, KE, and KER to AO will meet the criteria of 3Rs of the animal study and provide a quick and precise way to regulatory protection goals and decision-making. The overall weight of evidence (WoE) and level of certainty underlying the inference and extrapolation will in turn dictate the most suitable application of the AOP.

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6. Diagram/schematic figure

The pragmatic strategy to detect EDA of xenobiotics in food is to take a tier screening. Figure 4 showed the suggestion of flow chart for assessment of endocrine disruptors. Basically rat embryo culture could be the first screening method except for chemical structure-activity relationship.

Figure 4.

Suggestion of flow chart for assessment of endocrine disrupters.

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7. Conclusions

Penconazole, propiconazole, and triadimefon significantly induced the estrogen receptor expressions. It seems that WEC can be used as a robust method of endocrine-disrupting screening for estrogen receptors.

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Acknowledgments

The study was supported partly by the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, ROC (105AS-10.7.1-PI-P2), and partly by National Health Research Institutes, Zhunan, Miaoli County 35053, ROC (NHRI-106A1-PDCO-3416181).

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Conflicts of interest

The authors declare no conflicts of interest.

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Abbreviations

MRLmaximum residue level
EDCsendocrine-disrupting chemicals
OECDOrganization for Economic Co-operation and Development
OPPTSThe Office of Prevention, Pesticides, and Toxic Substances
rat WECwhole rat embryo culture
ARandrogen receptor
ERαestrogen receptor alpha
ERßestrogen receptor beta
TRαthyroid receptor alpha
TRßthyroid receptor beta
IHCimmunohistochemistry
WBwestern blot

References

  1. 1. Vinggaard AM, Hnida C, Breinholt V, Larsen JC. Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro. Toxicology In Vitro. 2000;14:227-234
  2. 2. Andersen HR, Cook SJ, Waldbillig D. Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicology and Applied Pharmacology. 2002;179:1-12
  3. 3. Kojima H, Katsura E, Takeuchi S, Niiyama K, Kobayashi K. Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. Environmental Health Perspectives. 2004;112:524-531
  4. 4. Lemaire G, Mnif W, Mauvais P, Balaguer P, Rahmani R. Activation of alpha- and beta-estrogen receptors by persistent pesticides in reporter cell lines. Life Sciences. 2006;79:1160-1169
  5. 5. Lemaire G, Mnif W, Pascussi JM, Pillon A, Rabenoelina F, Fenet H, et al. Identification of new human PXR ligands among pesticides using a stable reporter cell system. Toxicological Sciences. 2006;91:501-509
  6. 6. Kim DO, Lee SK, Jeon TW, Jin CH, Hyun SH, Kim EJ, et al. Role of metabolism in parathion-induced hepatotoxicity and immunotoxicity. Journal of Toxicology and Environmental Health, Part A. 2005;68:2187-2205
  7. 7. Singh AK. Acute effects of acephate and methamidophos and interleukin-1 on corticotropin-releasing factor (CRF) synthesis in and release from the hypothalamus in vitro. Comparative Biochemistry and Physiology, Part C: Toxicology & Pharmacology. 2002;132:9-24
  8. 8. Rollerová EE. Interaction of acetochlor with estrogen receptor in the rat uterus. Acetochlor—Possible endocrinemodulator? General Physiology and Biophysics. 2000;19:73-84
  9. 9. Crump D, Werry K, Veldhoen N, Van Aggelen G, Helbing CC. Exposure to the herbicide acetochlor alters thyroid hormone-dependent gene expression and metamorphosis in Xenopus laevis. Environmental Health Perspectives. 2002;110:1199-1205
  10. 10. Cocco P. On the rumors about the silent spring. Review of the scientific evidence linking occupational and environmental pesticide exposure to endocrine disruption health effects. Cadernos de Saúde Pública. 2002;18:379-402
  11. 11. Mikamo E, Harada S, Nishikawa J, Nishihara T. Endocrine disruptors induce cytochrome P450 by affecting transcriptional regulation via Pregnane X receptor. Toxicology and Applied Pharmacology. 2003;193:66-72
  12. 12. Klotz D, Arnold SF, McLachlan JA. Inhibition of 17 beta-estradiol and progesterone activity in human breast and endometrial cancer cells by carbamate insecticides. Life Sciences. 1997;60:1467-1475
  13. 13. Lemaire G, Terouanne B, Mauvais P, Michel S, Rahmani R. Effect of organochlorine pesticides on human androgen receptor activation in vitro. Toxicology and Applied Pharmacology. 2004;196:235-246
  14. 14. Cooper R, Stoker TE, Tyrey L, Goldman JM, McElroy WK. Atrazine disrupts the hypothalamic control of pituitary-ovarian function. Toxicological Sciences. 2000;53:297-307
  15. 15. Sanderson JT, Seinen W, Giesy JP, van den Berg M. 2-Chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells, a novel mechanism for estrogenicity? Toxicological Sciences. 2000;54:121-127
  16. 16. Hayes T, Tsui M, Hoang A, Haeffele C, Vonk A. Atrazine-induced hermaphroditism at 0.1 ppb in American leopard frogs (Rana pipiens); laboratory and field evidence. Environmental Health Perspectives. 2003;111:568-575
  17. 17. Thibaut R, Porte C. Effects of endocrine disrupters on sex steroid synthesis and metabolism pathways in fish. The Journal of Steroid Biochemistry and Molecular Biology. 2004;92:485-494
  18. 18. Morinaga H, Yanase T, Nomura M, Okabe T, Goto K, Harada N, et al. A benzimidazole fungicide; benomyl; and its metabolite; carbendazim; induce aromatase activity in a human ovarian granulose-like tumor cell line (KGN). Endocrinology. 2004;145:1860-1869
  19. 19. Kim IY, Shin JH, Kim HS, Lee SJ, Kang IH, Kim TS, et al. Assessing estrogenic activity of pyrethroid insecticides using in vitro combination assays. The Journal of Reproduction and Development. 2004;50:245-255
  20. 20. Trosken EE, Scholz K, Lutz RW, Volkel W, Zarn JA, Lutz WK. Comparative assessment of the inhibition of recombinant human CYP19 (aromatase) by azoles used in agriculture and as drugs for humans. Endocrine Research. 2004;30:387-394
  21. 21. Okubo T, Yokoyama Y, Kano K, Soya Y, Kano I. Estimation of estrogenic and antiestrogenic activities of selected pesticides by MCF-7 cell proliferation assay. Archives of Environmental Contamination and Toxicology. 2004;46:445-453
  22. 22. Goad R, Goad J, Atieh B, Gupta R. Carbofuran-induced endocrine disruption in adult male rats. Toxicology Mechanisms and Methods. 2004;14:233-239
  23. 23. Tessier D, Matsumura F. Increased ErbB-2 tyrosine kinase activity; MAPK phosphorylation; and cell proliferation in the prostate cancer cell line LNCaP following treatment by select pesticides. Toxicological Sciences. 2001;60:38-43
  24. 24. Tapiero HT, Nguyen BG, Tew KD. Estrogens and environmental estrogens. Biomedicine & Pharmacotherapy. 2002;56:36-44
  25. 25. Fang H, Tong W, Branham WS, Moland CL, Dial SL, Hong H, et al. Study of 202 natural; synthetic; and environmental chemicals for binding to the androgen receptor. Chemical Research in Toxicology. 2003;16:1338-1358
  26. 26. Vinggaard A, Breinholt V, Larsen JC. Screening of selected pesticides for oestrogen receptor activation in vitro. Food Additives and Contaminants. 1999;16:533-542
  27. 27. Kang HG, Jeong SH, Cho JH, Kim DG, Park JM, Cho MH. Chlropyrifos-methyl shows anti-androgenic activity without estrogenic activity in rats. Toxicology. 2004;199:219-230
  28. 28. Chen H, Xiao J, Hu G, Zhou J, Xiao H, Wang X. Estrogenicity of organophosphorus and pyrethroid pesticides. Journal of Toxicology and Environmental Health, Part A. 2002;65:1419-1435
  29. 29. McCarthy AR, Thomson BM, Shaw IC, Abella AD. Estrogenicity of pyrethroid insecticide metabolites. Journal of Environmental Monitoring. 2006;8:197-202
  30. 30. Bulayeva NN, Watson CS. Xenoestrogen-induced ERK-1 and ERK-2 activation via multiple membrane-initiated signaling pathways. Environmental Health Perspectives. 2004;112:1481-1487
  31. 31. Manabe M, Kanda S, Fukunaga K, Tsubura A, Nishiyama T. Evaluation of the estrogenic activities of some pesticides and their combinations using MtT/Se cell proliferation assay. International Journal of Hygiene and Environmental Health. 2006;209:413-421
  32. 32. Soto AM, Chung KL, Sonnenschein C. The pesticides endosulfan; toxaphene; and dieldrin have estrogenic effects on human estrogen-sensitive cells. Environmental Health Perspectives. 1994;102:380-383
  33. 33. Rawlings NC, Cook SJ, Waldbillig D. Effects of the pesticides carbofuran; chlorpyrifos; dimethoate; lindane; triallate; trifluralin; 2;4-D; and pentachlorophenol on the metabolic endocrine and reproductive endocrine system in ewes. Journal of Toxicology and Environmental Health, Part A. 1998;54:21-36
  34. 34. Mahjoubi-Samet A, Hamadi F, Soussia L, Fadhel G, Zeghal N. Dimethoate effects on thyroid function in suckling rats. Annales d’Endocrinologie. 2005;66:96-104
  35. 35. Hurst MRR, Sheahan DA. The potential for estrogenic effects of pesticides in headwater streams in the UK. Science of the Total Environment. 2003;301:87-96
  36. 36. Grunfeld HT, Bonefeld-Jorgensen EC. Effect of in vitro estrogenic pesticides on human estrogen receptor alpha and beta mRNA levels. Toxicology Letters. 2004;151:467-480
  37. 37. Tamura H, Yoshikawa H, Gaido KW, Ross SM, DeLisle RK, Welsh WJ, et al. Interaction of organophosphate pesticides and related compounds with the androgen receptor. Environmental Health Perspectives. 2003;111:545-552
  38. 38. Verslycke T. Testosterone and energy metabolism in the estuarine mysid Neomysis integer (Crustacea, Mysidacea) following exposure to endocrine disruptors. Environmental Toxicology and Chemistry. 2004;23:1289-1296
  39. 39. Garey J, Wolff MS. Estrogenic and antiprogestagenic activities of pyrethroid insecticides. Biochemical and Biophysical Research Communications. 1998;251:855-859
  40. 40. Eil CC, Nisula BC. The binding properties of pyrethroids to human skin fibroblast androgen receptors and to sex hormone binding globulin. Journal of Steroid Biochemistry. 1990;35:409-414
  41. 41. Chen HY, Liu R, He J, Song L, Bian Q, Xu L, et al. Effects of fenvalerate on progesterone production in cultured rat granulosa cells. Reproductive Toxicology. 2005;20:195-202
  42. 42. Richard S, Moslemi S, Sipahutar H, Benachour N, Seralini G-E. Differential effects of glyphosate and roundup on human placental cells and aromatase. Environmental Health Perspectives. 2005;113:716-720
  43. 43. Ralph JJ, Orgebin-Crist MC, Lareyre JJ, Nelson CC. Disruption of androgen regulation in the prostate by the environmental contaminant hexachlorobenzene. Environmental Health Perspectives. 2003;111:461-466
  44. 44. Verreault J, Skaare JU, Jenssen BM, Gabrielsen GW. Effects of organochlorine contaminants on thyroid hormone levels in Arctic breeding glaucous gulls; Larus hyperboreus. Environmental Health Perspectives. 2004;112:532-537
  45. 45. Beard AB, Rawlings NC. Thyroid function and effects on reproduction in ewes exposed to the organochlorine pesticides lindane or pentachlorophenol (PCP) from conception. Journal of Toxicology and Environmental Health, Part A. 1999;58:509-530
  46. 46. Oduma JA, Wango EO, Oduor-Okelo D, Makawiti DW, Odongo H. In vivo and in vitro effects of graded doses of the pesticide heptachlor on female sex steroid production in rats. Comparative Biochemistry and Physiology, Part C, Pharmacology, Toxicology & Endocrinology. 1995;111:191-196
  47. 47. Schmutzler C, Gotthardt I, Hofmann PJ, Radovic B, Kovacs G, Stemmler L, et al. Endocrine disruptors and the thyroid gland—A combined in vitro and in vivo analysis of potential new biomarkers. Environmental Health Perspectives. 2007;115:77-83
  48. 48. Ishihara A, Nishiyama N, Sugiyama S, Yamauchi K. The effect of endocrine disrupting chemicals on thyroid hormone binding to Japanese quail transthyretin and thyroid hormone receptor. General and Comparative Endocrinology. 2003;134:36-43
  49. 49. Porter W, Green SM, Debbink NL, Carlson I. Groundwater pesticides, interactive effects of low concentrations of carbamates aldicarb and methomyl and the triazine metribuzin on thyroxine and somatotropin levels in white rats. Journal of Toxicology and Environmental Health. 1993;40:15-34
  50. 50. Sonnenschein C, Soto AM. An updated review of environmental estrogen and androgen mimics and antagonists. The Journal of Steroid Biochemistry and Molecular Biology. 1998;65:143-150
  51. 51. Vinggaard AM, Hass U, Dalgaard M, Andersen HR, Bonefeld-Jørgensen E, Christiansen S, et al. Prochloraz, an imidazole fungicide with multiple mechanisms of action. International Journal of Andrology. 2006;29:186-192
  52. 52. Salazar KD, Schafer R, Barnett JB, Miller MR. Evidence for a novel endocrine disruptor, the pesticide propanil requires the ovaries and steroid synthesis to enhance humoral immunity. Toxicological Sciences. 2006;93:62-74
  53. 53. Kim SS, Kwack SJ, Lee RD, Lim KJ, Rhee GS, Seok JH, et al. Assessment of estrogenic and androgenic activities of tetramethrin in vitro and in vivo assays. Journal of Toxicology and Environmental Health, Part A. 2005;68:2277-2289
  54. 54. Nicolau GG. Circadian rhythms of RNA; DNA and protein in the rat thyroid; adrenal and testis in chronic pesticide exposure. III. Effects of the insecticides (dichlorvos and trichlorphon). Physiologie. 1983;20:93-101
  55. 55. Rovida C, Hartung T. Re-evaluation of animal numbers and costs for in vivo tests to accomplish REACH legislation requirements for chemicals: A report by the transatlantic think tank for toxicology (t(4)). ALTEX. 2009;26:187-208
  56. 56. Sogorb MA, Pamies D, de Lapuente J, Estevan C, Estúvez J, Vilanova E. An integrated approach for detecting embryotoxicity and developmental toxicity of environmental contaminants using in vitro alternative methods. Toxicology Letters. 2014;230:356-367
  57. 57. New DA, Coppola PT, Cockroft DL. Comparison of growth in vitro and in vivo of post-implantation rat embryos. Journal of Embryology and Experimental Morphology. 1976;36:133-144
  58. 58. Piersma AH. Validation of alternative methods for developmental toxicity testing. Toxicology Letters. 2004;149:147-153
  59. 59. Marotta F, Tiboni GM. Molecular aspects of azoles-induced teratogenesis. Expert Opinion on Drug Metabolism & Toxicology. 2010;6:461-482
  60. 60. Andrews JE, Ebron-McCoy M, Logsdon TR, Mole LM, Kavlock RJ, Rogers JM. Developmental toxicity of methanol in whole embryo culture: A comparative study with mouse and rat embryos. Toxicology. 1993;81:205-215
  61. 61. Dimopoulou M, Verhoef A, Pennings JLA, van Ravenzwaay B, Rietjens IMCM, Piersma AH. A transcriptomic approach for evaluating the relative potency and mechanism of action of azoles in the rat whole embryo culture. Toxicology. 2017;392:96-105
  62. 62. Brown NA, Fabro S. Quantitation of rat embryonic development in vitro: A morphological scoring system. Teratology. 1981;24:65-78
  63. 63. Robinson JF, van Beelen VA, Verhoef A, Renkens MF, Luijten M, van Herwijnen MH, et al. Embryotoxicant-specific transcriptomic responses in rat postimplantation whole-embryo culture. Toxicological Sciences. 2010;118:675-685. Erratum in: Toxicological Sciences. 2011;120:529
  64. 64. Robinson JF, Verhoef A, van Beelen VA, Pennings JL, Piersma AH. Dose-response analysis of phthalate effects on gene expression in rat whole embryo culture. Toxicology and Applied Pharmacology. 2012;264:32-41
  65. 65. Flick B, Talsness CE, Jäckh R, Buesen R, Klug S. Embryotoxic potential of N-methyl-pyrrolidone (NMP) and three of its metabolites using the rat whole embryo culture system. Toxicology and Applied Pharmacology. 2009;237:154-167
  66. 66. de Jong E, Barenys M, Hermsen SA, Verhoef A, Ossendorp BC, Bessems JG, et al. Comparison of the mouse embryonic stem cell test, the rat whole embryo culture and the zebrafish embryotoxicity test as alternative methods for developmental toxicity testing of six 1,2,4-triazoles. Toxicology and Applied Pharmacology. 2011;253:103-111
  67. 67. Robinson JF, Tonk EC, Verhoef A, Piersma AH. Triazole induced concentration-related gene signatures in rat whole embryo culture. Reproductive Toxicology. 2012;34:275-283

Written By

Shui-Yuan Lu, Pinpin Lin, Wei-Ren Tsai and Chen-Yi Weng

Submitted: 30 May 2018 Reviewed: 20 August 2018 Published: 05 November 2018