Methods to Test Endocrine Disruption in Drosophila melanogaster

Summary

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

Abstract

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-α-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Introduction

Human activities have been releasing into the environment a massive amount of chemicals, which represent a serious problem for organisms and for natural ecosystems¹. Of these pollutants, it is estimated that about 1,000 different chemicals may alter the normal balance of endocrine systems; according to this property, they are classified as endocrine disrupting chemicals (EDCs). Specifically, based on a recent definition by the Endocrine Society, the EDCs are “an exogenous chemical, or mixture of chemicals, that can interfere with any aspect of hormone action”². Over the last three decades, there has been grow....

Protocol

1. Food Preparation

1. For stock maintenance and for larval growth, use a cornmeal medium containing 3 % powdered yeast, 10 % sucrose, 9 % precooked cornmeal, 0.4 % agar, thereafter called Cornmeal medium (CM).
   1. Put 30 g of yeast into 100 mL of tap water, bring it to a boil and let it boil for 15 min.
   2. Separately, mix well 90 g of precooked cornmeal, 100 g of sugar, and 4 g of agar into 900 mL of tap water.
   3. Bring the solution to a boil, lower the heat and cook for 5 minutes stirring continuously.
   4. After 5 minutes, add the hot yeast solution and simmer for another 15 min.
   5. Turn off the heat s....

Results

In this section, key steps of the above protocols are reported in the form of simplified schemes. Given that flies tend to avoid unpalatable compounds, the first thing to do is to assay the taste of the selected EDC. This can be done by mixing a food coloring (for example, red food dye no. 40)³⁵ with the food supplemented with the selected EDC at various doses or with the solvent alone. Flies fed on these media are examined under a stereomicroscope and the food intake is estimated by their abdomin.......

Discussion

The fruit fly D. melanogaster has been extensively employed as an in vivo model system to investigate the potential effects of environmental EDCs such as DBP²⁸, BPA, 4-NP, 4-tert-OP²⁹, MP³⁰, EP^31,32, DEHP³³, and EE2³⁴. Several reasons have led its use as a model in this field of research. Apart from its undisputed advantages as a model .......

Materials

Name	Company	Catalog Number	Comments
17α-Ethinylestradiol	Sigma	E4876-1G
Agar for Drosophila medium	BIOSIGMA	789148
Bisphenol A	Sigma	239658-50G
Bisphenol AF	Sigma	90477-100MG
Cornmeal	CA' BIANCA
Diethyl ether	Sigma
Drosophila Vials	BIOSIGMA	789008	25x95 mm
Drosophila Vials	BIOSIGMA	789009	29x95 mm
Drosophila Vials	Kaltek	187	22X63
Embryo collection cage	Crafts		Plexiglass cylinder (12,5 x7 cm) with an open end and the other end closed by a rectangular base in which a slot allows the insertion of special trays for laying
Ethanol	FLUKA	2860
Etherizer	Crafts		cylindrical glass container with a cotton plug
Glass Bottle			250mL Bottles
Glass Vials	Microtech	ST 10024	FLAT BOTTOM TUBE 100X24
Hand blender Pimmy	Ariete		food processor
Instant Success yeast	ESKA		Powdered yeast
Laying tray	Crafts		plexiglass trays (11 x 2,6 cm) in wich to pour medium for laying
Methyl4-hydroxybenzoate	SIGMA	H5501
Petri Dish	Falcon	351016	60x5
Red dye no. 40	SIGMA	16035
Stereomicroscope with LED lights	Leica		S4E
Sucrose	HIMEDIA	MB025
Tomato sauce	Cirio