Name: assessment of chemical toxicity in adult drosophila melanogaster
Uploaded: 2023-03-24T14:00:00
Duration: 7 min 2 s
Description: Watch this Scientific Journal Video about Assessment of Chemical Toxicity in Adult Drosophila Melanogaster at JoVE.com

We thank our staff for help with the testing and optimization of this protocol: Ameya Belamkar, Marilyn Clark, Alexander Fitt, Emma Rose Gallant, Ethan Golditch, Matthew Lowe, Morgan Marsh, Kyle McClung, Andy Puga, Darcy Rose, Cameron Stockbridge, and Noelle Zolman. We also thank our colleagues from the Precision Toxicology Group, particularly our Exposure Group counterparts, for helping to identify the goals of the protocol.
This project received funding from the European Union&#39;s Horizon 2020 Research and Innovation program under Grant Agreement No. 965406. The work presented in this publication was performed as part of the ASPIS Cluster. This output reflects only the authors&#39; views, and the European Union cannot be held responsible for any use that may be made of the information contained therein. This publication was also made possible with support from the Indiana Clinical and Translational Sciences Institute, which is funded in part by Award Number UL1TR002529 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Portions of this project were supported by funds from Indiana University awarded to JRS and the PhyloTox consortium. JMH and EMP were supported by NIH award P40OD018537 to Bloomington Drosophila Stock Center.

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No conflicts of interest to disclose.

The fruit fly Drosophila melanogaster is emerging as a powerful system for NAMs16,18,19,21. By leveraging the unparalleled genetic resources available to the fly community, combined with recent advances in genomics and metabolomics, chemical safety studies using Drosophila are capable of quickly identifying the molecular mechanisms by which individual compounds interfere with metabolism, physiology, and cell signaling (for example, see39). This inexpensive protocol is designed to rapidly define dose-response curves and subsequently generate samples for RNA-seq and metabolomics analysis. Moreover, this flexible protocol can be adapted for use with any genotype and can accommodate many classes of chemicals.
A notable aspect of this protocol is the choice of liquid food used in the chemical exposure, which is based on a previous study, but differs from the solid media used by most toxicological studies of Drosophila18,22. This specific liquid media was selected to reflect the nutritional content of the standard, solid BDSC media that the flies are also fed in this protocol, to ensure the flies receive consistent nutrition. The simplicity of liquid feeding media has many advantages. Liquid media is easier to handle than solid food, which needs to be either melted and resolidified or reconstituted from powder. Liquid media also increases the system&#39;s throughput, ensures even chemical distribution throughout the feeding media, and decreases the time spent working with hazardous compounds. Additionally, the media does not require solutions to be heated, which facilitates the testing of volatile test compounds. Finally, because of the relatively few components included in the food solution, undesirable side reactions are minimized between the test chemical and other dietary components. The yeast used in the food is also inactive, further limiting the reactivity of the feeding medium. However, please note that the method is not suitable for testing developmental or larval toxicity.
Some of the materials used in the protocol can be substituted, such as using glass fly vials rather than polypropylene. However, the materials used were selected to be both inert and disposable to avoid unwanted chemical reactions between reagents and chemical exposures that could result from cleaning glassware.
The use of liquid food necessitates a vehicle for food delivery. Cellulose acetate filter paper was selected for this purpose due to its flexibility and inert nature28. Other researchers used similar protocols but with other vehicles, such as delicate task wipes or glass fiber filter29,30. The cellulose acetate filter paper suited these needs because it is an inert vehicle which can be cut to the ideal shape to fit it into the bottom of the fly vials without large gaps between the paper and vial wall, preventing death due to flies becoming stuck in media or the vehicle itself.
An important limitation of this system is that the maximum testable concentration of a chemical is tied to the solubility of the chemical. Non-water-soluble compounds require an additional solvent, which can lead to additional or synergistic effects with the chemical of interest. This can also create situations in which it is not possible to prepare stock solutions that are concentrated enough to achieve the desired endpoint in all organisms, therefore limiting analysis of the resulting data31. To address this, chemicals with low water solubility can be tested by adding up to 0.5% dimethyl sulfoxide to the food solution. Other solvents could be used as well, but additional research is needed for each solvent of interest to determine the maximum acceptable solvent concentration within the solution to maximize solubility while minimizing solvent effects on the organism.
Extensive characterization of the olfaction response in Drosophila has described how flies avoid consuming toxic compounds40,41, leading to reduced feeding on treated media. The blue dye assay addresses this phenomenon by allowing researchers to efficiently screen the feeding behaviors of the flies fed each concentration of experimental chemical42,43,44. The presence or absence of blue in the fly&#39;s gastrointestinal tract indicates if the fly has been eating the toxicant-containing medium. Although more sophisticated methods of assessing fly feeding behaviors exist, such as the Fly Liquid-Food Interaction Counter45, this qualitative method is better suited for higher-throughput screening.
A notable aspect of this protocol is that it has been optimized for a 48 h exposure period without the need to transfer flies or add additional liquid to the exposure vial. Using a humidity chamber and placing the chambers in an incubator kept at high humidity prevented the filter paper containing the feeding media from drying out during this timeframe. The protocol can be adapted for longer exposure durations, but the method must be adjusted to ensure that the filter paper does not become dry and cause significant changes in solution concentration or lethality due to desiccation.
Finally, an important characteristic of this protocol is that it can readily accommodate genetic variants, which allows researchers to utilize the vast array of genetic tools for Drosophila to expand these preliminary studies on wild-type organisms to better understand mechanisms of chemical action in vivo. In this regard, the protocol outlined above could be easily modified to complement a previously described JoVE&#160;protocol by Peterson and Long that allows for toxicological analysis of wild-caught flies18.
Because of the wide variety of previous studies on the toxicity of sodium arsenite in Drosophila32,33,34,35,36, Oregon-R flies were treated with this compound to demonstrate the efficacy of our system. Male flies exhibited an LD50 of 0.65 mM, and females exhibited an LD50 of 0.90 mM. This aligns with previous studies of sodium arsenite-treated adult Drosophila. For example, Goldstein and Babich37 found that 50% of flies (mixed sexes) died after 7 days of exposure to 0.5 mM NaAsO2. Although this is a slightly lower dose than was presently observed, the differences between their methods and this method (including the use of solid exposure media, a longer time scale, and mixed sexes) likely account for this difference. Importantly, both methods resulted in overall similar LD50 values.
Observations from experiments using this protocol can be used to find genetic and molecular targets for subsequent behavioral or mechanistic studies. The exposure method can also be used to treat Drosophila for sampling for metabolomics and proteomics, making this protocol well suited to the growing field of precision toxicology (modeled from the precision medicine field46). In this regard, exposed flies can be collected after step 8 for subsequent genomic and metabolomics analysis. Samples collected in step 8 can then be processed, as described by Li and Tennessen47, starting with step 3.
Ultimately, the data acquired from the experiments described above, as well as any subsequent metabolomics and proteomics data, would ideally be used in cross-species comparisons. As previously noted26, such cross-species studies are powerful and capable of determining how individual chemicals interfere with conserved biological pathways. Thus, the protocol described above can be used to find evolutionary commonalities in response to individual toxicants across phyla and help inform chemical safety regulation.

Humans are constantly exposed to chemicals from a variety of sources, including air1, food2, water3,4, medications5, cleaning agents6, personal care products7, industrial chemicals7, and building materials7. Moreover, thousands of new chemicals are introduced each year8, many of which are not properly vetted for health and environmental safety. This lack of adequate chemical safety testing stems in part from an over-reliance on mammalian models, such as mice and rats. While such rodent models are informative, chemical safety testing in these systems is expensive, time-consuming, and often causes unacceptable levels of suffering to the test animal9.
The financial and ethical burdens associated with mammalian chemical safety testing, as well as the time-consuming nature of mammalian studies, are major contributing factors to the paucity of data surrounding new chemicals. To address this issue, the U.S. Environmental Protection Agency (EPA), the European Chemicals Agency (ECHA), Health Canada, and other agencies are implementing measures that incorporate new approach methodologies (NAMs) into regulatory frameworks10, thus placing North American and European policy in line with international goals to replace, reduce, and refine the use of animals (the 3Rs principal)11,12,13,14. NAMs encompass a variety of assays primarily based on in vitro and in silico models that provide a mechanistic understanding of chemical toxicity instead of observing adversity inflicted on mammalian test species, thereby increasing the rate of data generation for chemical risk assessment while still producing high fidelity outputs15. However, these methods are not yet proven to safeguard against systemic toxicity, including the disruption of vital biological processes involving interorgan communication and endocrine signaling. Further, they cannot account for the bioaccumulation of chemicals within specific tissues, the ability of individual compounds to be absorbed and secreted, and the interplay between behavior and chemical exposure.
Due to the limitations of in vitro and computational models, the successful use of NAMs to reduce or replace mammalian models should also include invertebrate in vivo models, such as the fruit fly, Drosophila melanogaster. Previous studies in the fly have demonstrated that this organism is well suited for studying the conserved genetic pathways that protect animal cells against toxic molecules16,17,18,19,20,21,22. Moreover, the fly shows remarkable genetic similarity to humans, including functional homologs to over 65% of human diseases23,24,25 and an even greater conservation of important functional pathways26. These features, combined with their relatively short life cycle, low maintenance cost, and readily observable behavioral responses, make Drosophila well-suited for use as a toxicological model27,28,29,30. Moreover, flies have much higher throughput than rodent models and capture effects on metabolism, physiology, and hormone signaling that are not readily detectable by other non-organismal NAMs9.
The protocol described here represents a framework for testing the effects of chemical exposure on adult Drosophila. The method is designed to be efficient, inexpensive, and reproducible, while also minimizing the time researchers must be in contact with the test chemical and accommodating sample collection for metabolomics and other omics approaches. The protocol is optimized for testing a single chemical per experiment, but can easily accommodate other experimental parameters, such as varied solvents or combinations of chemicals.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 inch flower lever action craft punch</td>
			<td>Bira Craft</td>
			<td>HCP-115-024</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tubes&#160;</td>
			<td>VWR</td>
			<td>89039-666</td>
			<td>High-Performance Centrifuge Tubes with Flat or Plug Caps, Polypropylene, 15 mL</td>
		</tr>
		<tr>
			<td>2 ml Tubes&#160;</td>
			<td>VWR</td>
			<td>16466-044</td>
			<td>Micro Centrifuge Tube with Flat Screw-Cap, conical bottom</td>
		</tr>
		<tr>
			<td>5 ml Tubes&#160;</td>
			<td>VWR&#160;</td>
			<td>60818-576</td>
			<td>Culture Tubes, Plastic, with Dual-Position Caps</td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tubes&#160;</td>
			<td>Corning&#160;</td>
			<td>430290</td>
			<td>50 mL polypropylene centrifuge tubes, conical bottom with plug seal cap</td>
		</tr>
		<tr>
			<td>Benchmark Dose Software version 3.2</td>
			<td>U.S. Environmental Protection Agency</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cardboard trays</td>
			<td>Genesee Scientific flystuff</td>
			<td>32-122</td>
			<td>trays and dividers for narrow vials&#160;</td>
		</tr>
		<tr>
			<td>CO2 gas pads</td>
			<td>Genesee Scientific flystuff</td>
			<td>59-114</td>
			<td>FlyStuff flypad, CO2 anesthetizing apparatus&#160;</td>
		</tr>
		<tr>
			<td>Combitips advanced, 50 mL</td>
			<td>Eppendorf&#160;</td>
			<td>0030089693</td>
			<td>Combitips&#160;advanced, Biopur, 50&#160;mL, light gray, colorless tips</td>
		</tr>
		<tr>
			<td>Cotton balls</td>
			<td>Genesee Scientific flystuff</td>
			<td>51-101</td>
			<td>Cotton balls, large, fits narrow vials&#160;</td>
		</tr>
		<tr>
			<td>Delicate task wipes</td>
			<td>Kimtech</td>
			<td>34155</td>
			<td>Kimtech Science Kimwipes Delicate Task Wipes, 1 Ply / 8.2&#34; x 4.39&#34;</td>
		</tr>
		<tr>
			<td>Drosophila Vial Plugs, Cellulose Acetate (aka, Flugs)</td>
			<td>VWR</td>
			<td>89168-888</td>
			<td>Wide</td>
		</tr>
		<tr>
			<td>FD&#38;C Blue No. 1</td>
			<td>Spectrum Chemical</td>
			<td>FD110</td>
			<td>CAS number 3844-45-9</td>
		</tr>
		<tr>
			<td>Flies&#160;</td>
			<td>BDSC</td>
			<td>Stock #2057&#160;</td>
			<td>OregonR wildtype&#160;</td>
		</tr>
		<tr>
			<td>Gloves (nitrile)</td>
			<td>Kimtech</td>
			<td>55082/55081/55083</td>
			<td>Kimtech purple nitrile exam gloves, 5.9 mil, ambidextrous 9.5&#34;&#160;</td>
		</tr>
		<tr>
			<td>Grade 1 CHR cellulose chromatography paper</td>
			<td>Cytvia</td>
			<td>3001-917</td>
			<td>Sheet, 46 x 57 cm</td>
		</tr>
		<tr>
			<td>Mesh for humidity chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multipette / Repeater (X) stream&#160;</td>
			<td>Eppendorf&#160;</td>
			<td>022460811</td>
			<td>Repeater Xstream&#160;</td>
		</tr>
		<tr>
			<td>Plastic grate</td>
			<td>Plaskolite</td>
			<td>18469 (from lowes)</td>
			<td>Plaskolite 24 in x 48 in 7.85 sq ft louvered ceiling light panels, cut down to fit in rubbermaid tubs</td>
		</tr>
		<tr>
			<td>Plastic trays for glass vials</td>
			<td>Genesee Scientific flystuff</td>
			<td>59-207</td>
			<td>Narrow fly vial reload tray&#160;</td>
		</tr>
		<tr>
			<td>Polypropylene Drosophila Vial</td>
			<td>VWR</td>
			<td>75813-156</td>
			<td>Wide (28.5 mm)</td>
		</tr>
		<tr>
			<td>Rubbermaid tubs</td>
			<td>Rubbermaid</td>
			<td>3769017 (from Lowes)</td>
			<td>Rubbermaid Roughneck Tote 10 gallon 18&#34; L x 12&#34; W x 8 1/2&#34; H</td>
		</tr>
		<tr>
			<td>Sucrose ultra pure</td>
			<td>MP Biomedicals, Inc.</td>
			<td>821721</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube racks for wide-mouthed tubes</td>
			<td>Thermo scientific&#160;</td>
			<td>5970-0230</td>
			<td>Nalgene Unwire Test tube racks, for 30 mm tubes&#160;</td>
		</tr>
		<tr>
			<td>Water Purification System</td>
			<td>Millipore Milli-Q</td>
			<td>ZMQ560F01</td>
			<td>Millipore Milli-Q Biocel Water Purifier&#160;</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Acros Organics</td>
			<td>451120050</td>
			<td>CAS number 84604-16-0</td>
		</tr>
	</tbody>
</table>

assessment of chemical toxicity in adult drosophila melanogaster

Human industries generate hundreds of thousands of chemicals, many of which have not been adequately studied for environmental safety or effects on human health. This deficit of chemical safety information is exacerbated by current testing methods in mammals that are expensive, labor-intensive, and time-consuming. Recently, scientists and regulators have been working to develop new approach methodologies (NAMs) for chemical safety testing that are cheaper, more rapid, and reduce animal suffering. One of the key NAMs to emerge is the use of invertebrate organisms as replacements for mammalian models to elucidate conserved chemical modes of action across distantly related species, including humans. To advance these efforts, here, we describe a method that uses the fruit fly, Drosophila melanogaster,&#160;to assess chemical safety. The protocol describes a simple, rapid, and inexpensive procedure to measure the viability and feeding behavior of exposed adult flies. In addition, the protocol can be easily adapted to generate samples for genomic and metabolomic approaches. Overall, the protocol represents an important step forward in establishing Drosophila as a standard model for use in precision toxicology.

The fly has long served as a model in studies for determining sodium arsenite (NaAsO2) toxicity35,36,37,38. To demonstrate the efficacy of the protocol, male and female flies were exposed to NaAsO2, with the goal of comparing these results with earlier studies. Using the methodology described above, adult Oregon-R (BDSC stock #2057) males and females were exposed to a range of NaAsO2 concentrations (0, 0.01, 0.02, 0.1, 0.2, 1, and 2 mM) and scored for lethality 48 h after the start of exposure (Figure 1A,B).
The purpose of this initial analysis was to identify the approximate range of concentrations that would allow a more precise characterization of NaAsO2 toxicity. In subsequent experiments, concentrations were selected (0, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 5 mM) that more precisely defined the NaAsO2 dose-response curve (Figure 1C,D). Note that the resulting analysis examined several concentrations that induced 100% lethality. Data were analyzed using the Environmental Protection Agency&#39;s publicly available Benchmark Dose Software version 3.2.0.125. The data were modeled as &#34;dichotomous&#34; and the Dichotomous Hill model was used for subsequent analyses. Based on this model, the final LD10, LD25, and LD50 of male flies fed NaAsO2 were 0.30 mM, 0.50 mM, and 0.65 mM, respectively. For female flies, these values were slightly higher, with an LD10 of 0.30 mM, an LD25 of 0.65 mM, and an LD50 of 0.90 mM. Overall, the values obtained using this method are similar to those previously reported for arsenic toxicity in the Drosophila melanogaster35,36,37,38, thus validating the methodology.
In addition to the six replicates used to calculate the dose-response curve, male and female flies were also fed NaAsO2 exposure solutions that contained 1% FD&#38;C blue, which is easily visible in the digestive tract using light microscopy. Based on the presence of blue dye within the intestine of NaAsO2-fed flies, both male and female flies continued to feed 24 h after the beginning of the chemical exposure, regardless of the NaAsO2 concentration present within the liquid media (Figure 2). However, the ingested food was observed to be occasionally regurgitated at doses above 0.2 mM for females and 0.5 mM for males (Figure 2). These findings suggest that regurgitation could serve a key role in the Drosophila response to arsenic poisoning.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65029/65029fig01.jpg" /> 
Figure 1: Dose-response curves for male and female Drosophila treated with NaAsO2 for 48 h. All graphs show the estimated proportions of dead flies at each NaAsO2 concentration tested based on the Dichotomous Hill model. (A,B) A broad range of NaAsO2 concentrations were tested to approximate the dose at which each sex of fly begins to die. (A) shows the male data, and (B) shows the female data. N = 4 vials, with 20 flies per vial. (C,D) A narrower range of NaAsO2 concentrations was tested to determine precise doses at which 10%, 25%, and 50% of each sex of flies died. These doses are indicated to the right of each graph. (C) shows the male data, and (D) shows the female data. N = 6 vials, with 20 flies per vial. <a href="https://www.jove.com/files/ftp_upload/65029/65029fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65029/65029fig02.jpg" /> 
Figure 2: Representative results from the blue dye assay of male and female Drosophila treated with NaAsO2 for 24 h. Micrographs show flies fed increasing concentrations of NaAsO2. Row (A) shows male flies, and row (B) shows female flies, with the concentration of NaAsO2 increasing from left to right. The abdomen shows a small amount of blue near the thorax at low concentrations, indicating that exposure media entered the gut. At higher concentrations, blue dye begins to accumulate around the mouth, suggesting that exposure media is being regurgitated. The scale bar is 1 mm. <a href="https://www.jove.com/files/ftp_upload/65029/65029fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessment of Chemical Toxicity in Adult Drosophila Melanogaster at JoVE.com

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster

This protocol describes an efficient and inexpensive method that uses liquid media to assess the effects of chemical toxicants on the viability of adult Drosophila melanogaster.

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster

Human industries generate hundreds of thousands of chemicals, many of which have not been adequately studied for environmental safety or effects on human ...

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