Support by NSF (EPSCoR EPS-0814251) and NIH (1P20GM103653-01A1) to MKT and HSD, Delaware State University&#39;s institutional student support to MDM (Title III HBGI) and KRS (NIH-INBRE) is gratefully acknowledged. Thanks to the C. elegans Genetic Center (supported by NIH Office of Research Infrastructure Programs P40 OD010110) for C. elegans wild-type N2 strain.

Protocols given below have been standardized to test the effects of BPA exposure during early embryogenesis, and may be modified for use with BPS, BPF or other EDCs and toxicants on C. elegans embryos.
1. Material Setup
<ol>
	<li>Prepare 500 mL of Nematode Growth Media (NGM)18 media by dissolving 1.5 g of NaCl, 1.25 g of peptone, and 8.5 g of agar in water to 500 mL. After autoclaving (121 &#176;C, 15 PSI, 20 min), add 0.5 mL of cholesterol (5 mg/mL in ethanol), 0.5 mL of 1 M MgSO4, 0.5 mL of 1 M CaCl2 and 12.5 mL of 1 M potassium phosphate buffer, pH 7.4 (108.3 g of KH2PO4, 35.6 g of K2HPO4, water to 1 L).</li>
	<li>Prepare hypochlorite solution by mixing 25 mL of 1 N KOH, 4 mL of bleach, and 71 mL of water. Make this solution fresh.</li>
	<li>Prepare M9 Buffer by mixing 3 g of KH2PO4, 6 g of Na2HPO4, 5 g of NaCl, 5 mL of 1 M of MgSO4, and water to 1 L.</li>
	<li>Prepare S Buffer18 by mixing 129 mL of 0.02 M K2HPO4, 871 mL of KH2PO4, and 5.85 g of NaCl.</li>
	<li>Sterilize M9 and S buffers by autoclaving.</li>
	<li>Grow an overnight culture of Escherichia coli (OP 50 strain) in 20 mL of Luria Broth (LB).</li>
	<li>Dissolve BPA in 10% ethanol to make a 1 mM stock solution. Make subsequent dilutions of 0.1 &#181;M, 0.5 &#181;M, 1 &#181;M, 5 &#181;M and 10 &#181;M in S buffer. 
	NOTE: Dilution in the aqueous S buffer greatly reduces the concentration of ethanol; for example, at the highest BPA concentration described in this protocol (10 &#181;M), the concentration of ethanol is 0.1% v/v.</li>
</ol>
2. C. elegans Culturing and Synchronization
<ol>
	<li>Pour the NGM media into 100 mm plates (about 25 mL/plate) and allow it to solidify. Once solidified, seed the plates by spreading 150 &#181;L of E. coli OP50 from the liquid culture grown overnight. Incubate plates at 37 &#176;C overnight.</li>
	<li>The next day, transfer N2 worms onto the new plates by chunking an approximate slab of 1 cm square. Allow worms to grow for 3 days at 20 &#176;C until the plate is populated with well-fed mature adults containing visible embryos.</li>
	<li>To synchronize, wash each plate by pipetting up to 5 mL of M9 buffer across the plate. Transfer the liquid to a sterile 15 mL conical centrifuge tube. 
	NOTE: Synchronization is done to kill adult worms and collect embryos that are relatively the same stage.</li>
	<li>Centrifuge at 3,000 x g for 4 min at room temperature in order to obtain a loose pellet of adult worms.</li>
	<li>Using a 10 mL pipette, carefully remove the supernatant so as to leave the pellet intact. Add 5 mL of the hypochlorite solution and gently mix.</li>
	<li>Centrifuge at 3,000 x g at room temperature for 4 minutes.</li>
	<li>Remove supernatant with a 10 mL pipette and wash with 7 mL of M9 buffer. Repeat this step twice.</li>
</ol>
3. Exposing Worms to BPA
<ol>
	<li>After the last wash from step 2.7 above, suspend about 100 &#181;L of eggs with M9 buffer. Transfer about 50 &#181;L of eggs to 2 mL microfuge tubes already containing the appropriate concentration BPA diluted in S buffer. Adjust the amount of S-buffer to ensure the correct final concentration of BPA (0.0 &#181;M, 0.1 &#181;M, 0.5 &#181;M, 1.0 &#181;M, 5.0 &#181;M and 10 &#181;M) per tube. Table 1 illustrates one way to make these dilutions.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>For Final BPA Concentration</td>
			<td>Stock BPA (100 &#956;M)</td>
			<td>S Buffer</td>
			<td>Synchronized Worms</td>
		</tr>
		<tr>
			<td>0.1 &#956;M</td>
			<td>1 &#956;L</td>
			<td>949 &#956;L</td>
			<td>50 &#956;L</td>
		</tr>
		<tr>
			<td>0.5 &#956;M</td>
			<td>5 &#956;L</td>
			<td>945 &#956;L</td>
			<td>50 &#956;L</td>
		</tr>
		<tr>
			<td>1 &#956;M</td>
			<td>10 &#956;L</td>
			<td>940 &#956;L</td>
			<td>50 &#956;L</td>
		</tr>
		<tr>
			<td>5 &#956;M</td>
			<td>50 &#956;L</td>
			<td>900 &#956;L</td>
			<td>50 &#956;L</td>
		</tr>
		<tr>
			<td>10 &#956;M</td>
			<td>100 &#956;L</td>
			<td>850 &#956;L</td>
			<td>50 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 1: Stock BPA, S buffer and synchronized worms used to attain the desired concentrations used for our study.
<ol start="2">
	<li>Place tubes on a shaker and gently shake for 4 h at 20&#176;C, at 25 rpm for a rotary shaker or 25 tilts/min for a rocking shaker.</li>
	<li>After 4 h, place tubes in tube holders and allow worms to settle.</li>
	<li>Discard supernatant and transfer worms to seeded plates (NGM plates with OP50 E. coli). Make the liquid OP50 E. coli culture as stated in 1.5 and seed the plates as stated in 2.1.</li>
	<li>Let the worms grow for 60 h in 20 &#176;C. Examine plates every day for contamination. Contaminated NGM plates are usually discolored and accompanied by individual colonies. Check worms under the microscope for overcrowding. A lack of OP50 E. coli lawn and concentrated worms in small spots signify overcrowding.</li>
</ol>
4. Anterior Touch Habituation Assay
<ol>
	<li>Transfer approximately 10 synchronized young adult worms to new unseeded NGM plates using a fire-sterilized 30 G platinum wire pick. Leave worms undisturbed for 5 min to allow them time to acclimatize to the new plate.</li>
	<li>For the habituation response, use an eyebrow hair attached to the end of a wooden skewer or toothpick, and sterilize by dipping in 70% ethanol. Wipe with a clean lint-free tissue and wait for 1 min for ethanol to evaporate off. Gently touch the worm on the head (anterior of the pharyngeal bulb) using the eyebrow hair. Repeat the touches, allowing 10 s in between touches in order to allow the worm to recover.</li>
	<li>Continue to touch (allowing 10 s interstimulus intervals) until the worm no longer moves backwards. At this point, the worm has habituated to the stimulus. Record the number of touches required for the worm to habituate.</li>
	<li>Analyze the difference between the mean habituation rates of the treated and untreated controls using one-way ANOVA followed by Tukey&#39;s post hoc test.</li>
</ol>
5. Worm Fecundity Assay
<ol>
	<li>Transfer individual L3 worms to a fresh and seeded plate using a platinum pick. Make sure that only one worm is placed per plate. 
	NOTE: After the L3 larval stage, worms develop into L4 larval stage and adulthood. Picking them early is convenient in that they are large enough for the transfer of individual animals while ensuring that they have not already laid any eggs which otherwise would be missed in the count.</li>
	<li>Count the numbers of eggs laid every 12&#8211;24 h. After counting, transfer the parent worm to a new plate. Repeat until the worm stops laying eggs, typically after 5 days when incubated at 20 &#176;C. 
	NOTE: On average, a wild type worm can lay up to 300 eggs; therefore, transferring the parent to a new plate daily prevents overcrowding.</li>
	<li>Analyze the difference between the average number of eggs laid by the treated and untreated controls using one-way ANOVA followed by Tukey&#39;s post hoc test.</li>
</ol>

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The authors declare that they have nothing to disclose.

Phases of rapid growth and development such as embryogenesis are particularly vulnerable to the detrimental effects of various endocrine-disrupting compounds, including BPA. We provide detailed protocols for studying the effects of exposure to BPA or other toxicants in the C. elegans invertebrate model, which allows convenient titration of a range of concentrations to assess its effect on embryo viability (Figure 2). Follow up behavioral experiments on surviving adults that develop from embryos that were exposed to low doses of BPA allow assaying long-term effects on neuronal function (Figure 3). The central idea of this paper is to provide detailed protocols for exposing embryos to various toxicants during the early embryogenesis period in the C. elegans model; the first 4 h window of its 11.5 hour embryonic development period (at 20 &#176;C) corresponds to the proliferation period, which is followed by organogenesis. Towards this end, we have provided validation of the end points through fecundity and non-associative learning assays. While the mechanistic basis of the action of BPA and other bisphenols is the holy grail towards which research in this field is aimed, we have not attempt to offer a mechanistic explanation for the action of BPA in this methodology focused article. However, we would like to point out that the C. elegans model offers an ideal system to further dissect the mechanism of action. For example, in future work on deciphering mechanisms of BPA action, C. elegans mutants can be generated and screened for a resistance to the effects of BPA and thus pave the way for deciphering the pathway. Additionally, our companion paper deals with the effect of BPA at the neural network synchrony level in vertebrates, providing another avenue to understand the basis for investigating potential mechanisms for toxicant effects19.
In the methodology detailed in this paper, some of the critical steps that should be executed extra carefully include the following: Choosing a plate with well-fed C. elegans adults with mature eggs visible under the dissection microscope is crucial, and failing to follow this step will result in an insufficient sample size of embryos for the EDC exposure experiment. It is also important to make sure that the hypochlorite solution is made fresh to avoid obtaining an unsynchronized population that will interfere with the ability to perform BPA exposure during early embryogenesis. Washing pellets with M9 (after hypochlorite exposure) is also a very important step. If not done properly, the high concentration of bleach might result in dead embryos. Additionally, embryos should be transferred to seeded NGM plates after 4 h. If left in the BPA solution for an extended period of time, the embryos are likely to develop into the long-lived dauer larvae. This will significantly interfere with both fecundity assay and behavioral assays. If any of these steps is missed, it is advisable to start the procedure from the very beginning. If for any reason, the NGM plate with worms is contaminated or does not have enough food, it will add stress on the worms thereby skewing the data collected. Such plates should be discarded and not be used in the experiment.
Previously, a remarkable study successfully used the C. elegans model to demonstrate that chromosome abnormalities caused by BPA result due to the breakdown of DNA repair machinery in the germline14. However, it is noteworthy that the above study design had used continuous BPA exposure of C. elegans to high doses of BPA throughout its lifespan. Our methodology was designed to study low doses of BPA exposure during early embryogenesis, so as to assay its effects on embryonic viability and subtler long-term effects on the survivors. To our knowledge, no methodology has been developed to expose early stage embryos to very low doses of BPA limited to a specified period of time; thus, making the method presented here novel.
In addition, protocols provided here can be easily adapted to study the effects of other EDCs during the proliferation phase of embryogenesis. However, in order to study late embryonic stages, the protocol will require key modifications to the critical time window of development under focus. Furthermore, our protocol will not be suitable for experiments requiring longer times of toxicant exposure, as it opens up the possibility of the alternate development pathway resulting in the long-lived dauer arrest at the second molting cycle, which is resistant to harsh conditions20. Further modifications and refinements will be needed for longer exposure times through supplement with a food source that does not have potential to metabolize the toxicant or EDC e.g. autoclaved E. coli. In conclusion, our methodology can be utilized to assess the effects of various endocrine disrupting chemicals on the fecundity and on neuronal function in the invertebrate model system C. elegans.

Exposure to environmental toxicants, particularly compounds those that tend to interfere with development, has been under careful scientific scrutiny in recent years. More than a thousand chemicals in everyday use are classified as endocrine disrupting compounds (EDC)1. Periods of rapid growth and development, which include embryonic, infant and childhood stages, have been noted to be particularly vulnerable to the deleterious effects to even low-dose EDC. Their effect have been shown to cause reproductive and neurodevelopmental disorders2. Per the US Environmental Protection Agency and US National Toxicology Program panel guidelines, a low dose may be defined as any dose below the level of one that has been reported to cause an observable biological change or damage3. Besides low-dose effects of individual EDCs, mixtures of various EDCs found at low concentrations in the environment have the potential to cause substantive cumulative effects4.
Bisphenol A (BPA or 4,4&#39;-(propane-2,2-diyl)) is a polymerizing agent found in commonly used items such as water bottles, store receipts, dental sealants, and the linings of beverage and food cans5. Due to its structural similarity to 17-&#946; Estradiol (E2) and its affinity to the ER&#945; and ER&#946; estrogen receptors, BPA has been classified as an endocrine disrupting chemical (EDC)5,6.Although weaker, BPA&#39;s affinity to estrogen receptors has been shown to affect the reproduction system of both sexes and disrupt neural functions at doses that are considered safe7,8. Changes in DNA methylation via epigenetically regulated mechanisms have been observed to cause long-term neuronal defects in mice exposed to BPA9. Specifically, BPA has also been implicated as a possible culprit for increased rates of hyperactivity, attention deficiency and increased sensitivity to drugs due to an increase seen in D1 dopamine receptors in the mouse limbic forebrain after chronic exposure9,10. Significant evidence on the deleterious effects of EDCs on human health is based on correlation studies focusing mainly on chronic exposure of populations to environmental toxicants even at low doses5; however, limitations in inferences from human studies and in manipulating experimental controls have been accepted while addressing criticisms of unsubstantiated hype11,12.
Due to the conservation of Caenorhabditis elegans&#39; genes with respect to mammals&#39;, including its steroid hormone-receptor genes, researchers have utilized this genetically tractable lab model to unravel the functional and mechanistic effects of EDCs13. Experiments with C. elegans have shown that these compounds such as BPA and BPS can cause apoptosis, embryonic lethality, disruption in the double-stranded DNA break repair mechanisms and neural function14,15,16.
Our lab has previously shown that even low-dose exposure limited to early embryogenesis leads to lowered fecundity with behavioral deficits in the surviving adults15. Habituation to a repeated stimulus is a form of non-associative learning in model systems, including C. elegans, and our methodology uses this form of non-associative learning as a behavioral output to assay the long-term effects of embryonic exposure to the toxicants17 Specifically, we provide detailed protocols for studying BPA exposure on C. elegans, including its immediate effects on embryonic lethality and long-term effects on adult behavior, with an overall schematic depicted in Figure 1. Representative results from fecundity and non-associative learning assays are provided to highlight the effectiveness of our methodology.

<table><tbody><tr><td>NaCl</td><td>Fisher</td><td>7647-14-5</td><td></td></tr><tr><td>Peptone</td><td>Fisher</td><td>S25761B</td><td></td></tr><tr><td>Agar</td><td>Fisher</td><td>BP1423-500</td><td></td></tr><tr><td>Cholesterol</td><td>Alfa Aesar</td><td>A11470</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>7487-88-9</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>C79-500</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>P286-1</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>7758-11--4</td><td></td></tr><tr><td>KOH</td><td>Fisher</td><td>1310-58-3</td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td>n/a</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>BP332-500</td><td></td></tr><tr><td>LB</td><td>Fisher</td><td>BP1426-500</td><td></td></tr><tr><td>BPA</td><td>Sigma-Aldrich</td><td>239658-250g</td><td></td></tr><tr><td>BPS</td><td>Sigma-Aldrich</td><td>103039-100g</td><td></td></tr><tr><td>EtOH</td><td>Sigma-Aldrich</td><td>64-17-5</td><td></td></tr><tr><td>E.coli OP50</td><td>CGC</td><td></td><td>Repository at U of Minnesota</td></tr><tr><td>N2 (Wild-type C. elegans) worms</td><td>CGC</td><td></td><td>Repository at U of Minnesota</td></tr><tr><td>Platinum wire pick</td><td>Genesee Scientific</td><td>59-AWP</td><td></td></tr><tr><td>Petri plates</td><td>Fisher</td><td>07-202-011</td><td></td></tr><tr><td>Dissection Microscope</td><td>AmScope</td><td>SM-2TYY</td><td></td></tr></tbody></table>

long-term behavioral and reproductive consequences of embryonic exposure to low-dose toxicants

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use products. Based on their chemical structure and estradiol-like biological properties, they have been classified as endocrine disrupting compounds (EDC). Long-term exposure to EDCs, even at low doses, has been linked to various health defects including cancer, behavioral disorders and infertility, with greater vulnerability indicated during early developmental periods. Cellular and molecular studies with the genetically tractable nematode model Caenorhabditis elegans have demonstrated that exposure to BPA causes apoptosis, embryonic lethality and disruption in the DNA repair mechanisms. We have previously reported that exposure of C. elegans embryos to low doses of different bisphenols decreases fecundity. In addition, we have shown that the effects of exposure during the very early stages of development persist into adulthood as assayed by quantifying habituation behavior, a form of non-associative learning. Here, we provide detailed protocols for embryonic exposure to low-dose EDCs as well as the associated fecundity and anterior touch habituation assays, along with representative results.

Earlier studies with C. elegans have reported that continual exposure to high BPA concentrations (&#8805;1 mM) throughout embryo development and adulthood decreases fecundity14. Subsequent studies reported from our lab have shown that embryos that have been exposed to BPA for a limited period of 4 h in the beginning stages of their development showed a decrease in the number of viable eggs laid as adults15 (Figure 2). Two key features of the methodology were that (i) the BPA concentrations used were significantly lower (0.1 &#181;M to 10 &#181;M range) and (ii) the exposure was limited to the early embryonic period. Besides decreased fecundity even at lower doses, our habituation assays revealed that worms exposed to BPA as embryos required more stimuli to become habituated when compared to worms exposed to vehicle alone15 (Figure 3). These effects are noteworthy in that they are based on low-dose BPA exposure, following the rationale that subtler effects on neuronal function that may not be morphologically apparent are likely to be discernible through behavioral changes. In short, the representative results presented here underscore the fact that the detrimental effects of BPA exposure to an embryo impacts its development, including neuronal function that can be assessed through adult behavioral assays in C. elegans. Protocols provided here can be used for testing low-dose as well as long-term effects of other potential toxicants including endocrine disruptive compounds.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56771/56771fig1.jpg" /> 
Figure 1: Schematic representation of the experimental design. Mature worms were collected and exposed to hypochlorite solution in order to collect synchronized embryos. The embryos were then exposed to different concentrations of BPA for 4 h. The exposed embryos were transferred onto seeded NGM plates, where they were allowed to grow for 60 h at 20 &#176;C. In order to test the effectiveness of exposure during the early stages of development, the L4 worms were transferred to individual plates and tested for fecundity, and young adults were tested with anterior touch habituation. <a href="https://cloudflare.jove.com/files/ftp_upload/56771/56771fig1large.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/56771/56771fig2.jpg" /> 
Figure 2: Fecundity is lowered by BPA. Exposure to 1 &#181;M and higher concentrations of BPA significantly decreased the number of eggs laid compared to the control (represented by the horizontal line; n = 10, *p &#60;0.05). Error bars denote SEM. Analysis done using ANOVA, Tukey&#39;s post hoc test. This figure has been modified from Mersha et al., 201515. <a href="https://cloudflare.jove.com/files/ftp_upload/56771/56771fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56771/56771fig3.jpg" /> 
Figure 3: Long-term effects of embryonic BPA exposure are reflected in adult behavior. Embryos exposed to BPA concentrations as low as 0.1 &#181;M affects non-associative anterior touch habituation in adults (n = 60, *p &#60;0.05). Error bars denote SEM; ANOVA, Tukey&#39;s post hoc test. This figure has been modified from Mersha et al., 201515. <a href="https://cloudflare.jove.com/files/ftp_upload/56771/56771fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants at JoVE.com

Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

Exposure to environmental toxicants can acutely impact development with long-term effects. Detailed protocols are provided to illustrate a strategy using an effective lab model to study the effect of early embryonic exposure to bisphenol A. We provide fecundity and behavioral assays to monitor the effectiveness of our toxicant exposure bio-assays.

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use ...

long-term-behavioral-reproductive-consequences-embryonic-exposure-to

Research

JoVE Journal

Developmental Biology

6.0K Views.  Johns Hopkins School of Medicine. Blootstelling aan toxische stoffen in het milieu kan acuut ontwikkeling met effecten op lange termijn beïnvloeden. Gedetailleerde protocollen worden geleverd om te illustreren een strategie met behulp van een effectieve lab-model om te bestuderen van het effect van vroege embryonale blootstelling aan Bisfenol A. Wij bieden vruchtbaarheid en gedrags tests om de doelmatigheid van onze toxische blootstelling bio-tests.

Video: Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE1, and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

The goal of the protocol presented here is to describe procedures to expose rats to moderate levels of alcohol during prenatal brain development and to quantify resulting alterations in social behavior during adulthood.

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several ...

Behavior

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

Environment

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday products. They are classified as endocrine disrupting compounds (EDC) with estradiol-like properties. Long-term exposure to EDCs, even at low doses, has been linked with various health defects including cancer, behavioral disorders, and infertility, with greater vulnerability during early developmental periods. To study the effects of BPA on the development of neuronal function, we used an in vitro neuronal network derived from the early chick embryonic brain as a model. We found that exposure to BPA affected the development of network activity, specifically spiking activity and synchronization. A change in network activity is the crucial link between the molecular target of a drug or compound and its effect on behavioral outcome. Multi-electrode arrays are increasingly becoming useful tools to study the effects of drugs on network activity in vitro. There are several systems available in the market and, although there are variations in the number of electrodes, the type and quality of the electrode array and the analysis software, the basic underlying principles, and the data obtained is the same across the different systems. Although currently limited to analysis of two-dimensional in vitro cultures, these MEA systems are being improved to enable in vivo network activity in brain slices. Here, we provide a detailed protocol for embryonic exposure and recording neuronal network activity and synchrony, along with representative results.

Exposure to environmental toxins can acutely impact developing embryos. Endocrine disrupting chemicals such as bisphenols are known to adversely affect the nervous system. Here we describe a protocol using an in vitro vertebrate (chick embryo) neuronal network model to study the functional impact of toxin exposure on early embryos.

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues and organs. Ecotoxicological effects of microplastics on the development of bird embryos are not known. The bird egg is a complete development and nutrition system, and the entire embryo development occurs in the eggshell. Therefore, a direct record of bird embryo development under the stress of pollutants such as microplastics is highly limited by the opaque eggshell in traditional hatching. In this study, the effects of microplastics on quail embryo development were visually monitored by hatching without an eggshell. The main steps include the cleaning and disinfection of fertilized eggs, the incubation before exposure, the short-term incubation after exposure, and the sample extraction. The results show that compared with the control group, the wet weight and body length of the microplastics-exposed group displayed a statistical difference and the liver proportion of the whole exposed group significantly increased. Additionally, we evaluated external factors that affect the incubation: temperature, humidity, egg rotation angle, and other conditions. This experimental method provides valuable information on the ecotoxicology of microplastics and a novel way to study the adverse effects of pollutants on the development of embryos.

This paper introduces a method of hatching without using an eggshell for toxicological studies of particle pollutants such as microplastics.

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues ...

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

Medicine

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is well-defined, and it is relatively easy to achieve precise and timely exposure via multiple methods. Moreover, the process of heart development in chicken embryos is similar to mammals, also resulting in a four-chambered heart, making it a valuable alternative model in the assessment of developmental cardiotoxicities. In our lab, the chicken embryo model is routinely used in the assessment of developmental cardiotoxicities following exposure to various environmental pollutants, including per- and polyfluoroalkyl substances (PFAS), particulate matter (PMs), diesel exhaust (DE) and nano materials. The exposure time can be freely selected based on the need, from the beginning of development (embryonic day 0, ED0) all the way to the day prior to hatch. The major exposure methods include air-cell injection, direct microinjection, and air-cell inhalation (originally developed in our lab), and the currently available endpoints include cardiac function (electrocardiography), morphology (histological assessments) and molecular biological assessments (immunohistochemistry, qRT-PCR, western blotting, etc.). Of course, the chicken embryo model has its own limitations, such as limited availability of antibodies. Nevertheless, with more laboratories starting to utilize this model, it can be used to make significant contributions to the study of developmental cardiotoxicities.

Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Biology

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

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